Ccr3 modulation in the treatment of aging-associated impairments, and compositions for practicing the same

ABSTRACT

Methods of treating an adult mammal for an aging-associated impairment are provided. Aspects of the methods include modulating CCR3, e.g., by modulating eotaxin-1/CCR3 interaction, in the mammal in a manner sufficient to treat the mammal for the aging-associated impairment. A variety of aging-associated impairments may be treated by practice of the methods, which impairments include cognitive impairments.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. application Ser. No. 14/991,813filed on Jan. 8, 2016; which application is a continuation-in-partapplication of U.S. application Ser. No. 14/280,939 filed May 19, 2014;which application is a Continuation Application of U.S. application Ser.No. 13/575,437, filed on Oct. 9, 2012 and now abandoned; whichapplication is a 35 U.S.C. § 371 National Phase Entry Application ofInternational Application Serial No. PCT/US2011/022916, filed Jan. 28,2011, which designates the United States, and which claims benefit under35 U.S.C. § 119(e) of the U.S. Provisional Application Ser. No.61/298,998, filed on Jan. 28, 2010; the disclosures of whichapplications are herein incorporated by reference in their entirety.

GOVERNMENT RIGHTS

This invention was made with Government support under contract AG027505and OD000392 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

INTRODUCTION

Aging in an organism is accompanied by an accumulation of changes overtime. In the nervous system, aging is accompanied by structural andneurophysiological changes that drive cognitive decline andsusceptibility to degenerative disorders in healthy individuals. (Hedden& Gabrieli, “Insights into the ageing mind: a view from cognitiveneuroscience,” Nat. Rev. Neurosci. (2004) 5: 87-96; Raz et al.,“Neuroanatomical correlates of cognitive aging: evidence from structuralmagnetic resonance imaging,” Neuropsychology (1998) 12:95-114; Mattson &Magnus, “Ageing and neuronal vulnerability,” Nat. Rev. Neurosci. (2006)7: 278-294; and Rapp & Heindel, “Memory systems in normal andpathological aging,” Curr. Opin. Neurol. (1994) 7:294-298). Included inthese changes are synapse loss and the loss of neuronal function thatresults. Thus, although significant neuronal death is typically notobserved during the natural aging process, neurons in the aging brainare vulnerable to sub-lethal age-related alterations in structure,synaptic integrity, and molecular processing at the synapse, all ofwhich impair cognitive function.

In addition to the normal synapse loss during natural aging, synapseloss is an early pathological event common to many neurodegenerativeconditions, and is the best correlate to the neuronal and cognitiveimpairment associated with these conditions. Indeed, aging remains thesingle most dominant risk factor for dementia-related neurodegenerativediseases such as Alzheimer's disease (AD) (Bishop et al., “Neuralmechanisms of ageing and cognitive decline,” Nature (2010) 464: 529-535(2010); Hedden & Gabrieli, “Insights into the ageing mind: a view fromcognitive neuroscience,” Nat. Rev. Neurosci. (2004) 5:87-96; Mattson &Magnus, “Ageing and neuronal vulnerability,” Nat. Rev. Neurosci. (2006)7:278-294).

As human lifespan increases, a greater fraction of the populationsuffers from aging-associated cognitive impairments, making it crucialto elucidate means by which to maintain cognitive integrity byprotecting against, or even counteracting, the effects of aging (Hebertet al., “Alzheimer disease in the US population: prevalence estimatesusing the 2000 census,” Arch. Neurol. (2003) 60:1119-1122; Bishop etal., “Neural mechanisms of ageing and cognitive decline,” Nature (2010)464:529-535).

SUMMARY

Methods of treating an adult mammal for an aging-associated impairmentare provided. Aspects of the methods include modulating CCR3, e.g., viamodulation of eotaxin-1/CCR3 interaction, in the mammal in a mannersufficient to treat the mammal for the aging-associated impairment. Avariety of aging-associated impairments may be treated by practice ofthe methods, which impairments include cognitive impairments.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1E show that heterochronic parabiosis reduces adultneurogenesis in young animals while increasing neurogenesis in agedmice. FIG. 1A shows a schematic of the three combinations of mice usedin isochronic and heterochronic pairings. FIG. 1B shows quantificationof neurogenesis in the young DG after parabiosis. Data are from 12 micefor isochronic and 10 mice for heterochronic groups (5-7 sections permouse). FIG. 10 shows quantification of neurogenesis in the old DG afterparabiosis. Data are from 6 mice for isochronic and 12 mice forheterochronic groups (5-7 sections per mouse; **, P<0.01). e, Highmagnification view of neurite arbors from Doublecortin-positive neuronsfrom young (scale bar: 50 μm) and old (scale bar: 25 μm) parabioticpairings. FIG. 1D shows quantification of average neurite length fromyoung isochronic and heterochronic parabionts. The length of the longestvisible neurite was measured in 250 neurons (measured in random fieldsacross 5 sections per mouse). FIG. 1E shows quantification of averageneurite length from old isochronic and heterochronic parabionts asdescribed for young mice. Mean+SEM; *, P<0.05; **, P<0.01 t-test.

FIGS. 2A-2E show that exposure of a young adult brain to an old systemicenvironment decreases synaptic plasticity and impairs spatial learningand memory. FIG. 2A shows quantification of neurogenesis in the young DGafter plasma injection. Data are from 7-8 mice per group (5-7 sectionsper mouse). FIGS. 2B and 2C show experiments where synaptic plasticityof young isochronic and heterochronic parabionts was examined after fiveweeks of parabiotic pairing in hippocampal slices by extracellularelectrophysiological recordings using a long-term potentiation (LTP)paradigm. FIG. 2B shows representative electrophysiological profilescollected from individual young (3 months) isochronic and heterochronicparabionts during LTP recordings from the DG. FIG. 2C shows that LTPlevels recorded from the DG were lower in the hippocampus of youngheterochronic (100.6±34.3%) versus young isochronic (168.5±15.8%)parabionts following 40 minutes after induction. Data are from 4-5 miceper group. FIGS. 2D and 2E show how spatial learning and memory wasassessed using the radial arm water maze (RAWM) paradigm in young (3months) adult male mice injected intravenously with plasma isolated fromyoung (3-4 months) and old (18-20 months) mice every three days for 24days. FIG. 2D shows a schematic of the RAWM paradigm. The goal armlocation containing the platform remains constant, while the start armis changed during each trial. On day one during the training phase, miceare trained for 15 trials, with trials alternating between visible(white) and hidden (shaded) platform. On day two during the testingphase, mice are tested for 15 trials with the hidden (shaded) platform.Entry into an incorrect arm is scored as an error, and errors areaveraged over training blocks (three consecutive trials). FIG. 2E showshow learning and memory deficits were quantified as the number of entryarm errors made prior to finding the target platform. Data are from 7-8mice per group. Mean±SEM; *, P<0.05; **, P<0.01; t-test (2A), ANOVA,Tukey's post-hoc test (2E).

FIGS. 3A-3I show that systemic chemokine levels increase during normalaging and heterochronic parabiosis and correlate with the age-dependentdecrease in neurogenesis. FIG. 3A shows a Venn diagram outlining theresults from the normal aging and parabiosis proteomic screens. Theseventeen blood borne factors whose levels increased with aging andcorrelated strongest with the age-related decline in neurogenesis areshown in left side circle, the fourteen blood borne factors thatincreased between young isochronic and young heterochronic parabiontsare shown in right side circle, and the five factors elevated in bothscreens are shown in the intersection in light grey area. (5-6 animalsper age group were used) FIGS. 3B-3E show changes in plasmaconcentrations for CCL2 (3B, 3D) and CCL11 (3C, 3E) with age (3B, 3C)and from an independent proteomic screen in young heterochronicparabionts pre- and post-parabiotic pairing (3D, 3E). FIGS. 3F-3I showchanges in concentrations for CCL2 (3F, 3H) and CCL11 (3G, 3I) inhealthy, cognitively normal human subjects in plasma with age (3F, 3G)and in CSF between young (20-45 years) and old (65-90 years) (3H, 3I).Dot plots with mean; *, P<0.05; **, P<0.01; ***, P<0.001 t-test (c,d),ANOVA, Tukey's post-hoc test (3A, 3B), and Mann-Whitney U Test (3H, 3I).

FIGS. 4A-4G show that systemic exposure to the age-related chemokineCCL11 inhibits neurogenesis and impairs spatial learning and memory inyoung adult animals. FIG. 4A shows an experiment where Dcx-luc reportermice (2-3 months) were injected with either recombinant murine CCL11 orPBS (vehicle) every other day for four days (7 mice per group).Bioluminescence was recorded in living mice at days zero and four, andrepresentative images are shown for each treatment group. FIG. 4B showsresults when bioluminescence was quantified as photons/s/cm2/steradianand differences expressed as changes in fold-induction between day zeroand four. FIG. 4C shows quantification of neurogenesis in the DG aftersystemic drug administration after an independent cohort of 3-month-oldwild type male mice was injected intraperitoneally with recombinantmurine CCL11 or vehicle alone, and in combination with an anti-CCL11neutralizing antibody or an isotype control antibody four times over tendays (6-10 mice per group). FIG. 4D shows quantification of the relativenumber of BrdU and NeuN double positive cells compared to the totalnumber of BrdU positive cells in the DG mice that were systemicallyadministered with either recombinant murine CCL11 or vehicle alone fromthe group above were injected with BrdU daily for three days prior tosacrifice. FIGS. 4E-4F show quantification of neurogenesis in the DGafter systemic and stereotaxic drug administration. Data are from 3-10young adult mice (2-3 months) per group (5 sections per mouse) afteryoung adult mice were given unilateral stereotaxic injections of eitheranti-CCL11 neutralizing antibody or an isotype control antibody followedby systemic injections with either recombinant CCL11 or PBS. FIG. 4Gshows how spatial learning and memory was assessed using the RAWMparadigm in young adult male mice (3 months) injected with recombinantmurine CCL11 or PBS (vehicle) every three days for five weeks. Cognitivedeficits were quantified as the number of entry arm errors made prior tofinding the target platform. All the histological and behavioralassessments were carried out by investigators blinded to the treatmentof the mice. Data is represented as Mean±SEM; *, P<0.05; **, P<0.01;t-test (4B, 4D, 4E, 4F), ANOVA, Dunnett's or Tukey's post-hoc test (4C,4G).

FIGS. 5A-5D show that adult neurogenesis decreases as neuroinflammationincreases in the DG during aging. We performed an immunohistochemicaldetection of newly differentiated Doublecortin (Dcx)-positive neurons,long-term BrdU-retaining cells (arrowheads), CD68-positive activatedmicroglia, and GFAP-positive astrocytes in the DG of the hippocampusfrom adult mice at 6 and 18 months of age. FIGS. 5A-5D showquantification of age-related cellular changes in the adult DG. Data arefrom 5-10 mice per age group (5-7 sections per mouse), each dotrepresents the mean number per mouse Animals were given 6 days of BrdUinjections and euthanized 21 days following the last injection. FIG. 5Cshows age-related increase of relative immunoreactivity to CD68, amarker for microglia activation. FIG. 5D shows that GFAP reactivity didnot significantly change with age. Dot plots with mean; ***, P<0.001,ANOVA, Dunnett's post-hoc test.

FIGS. 6A-6B show that synaptic plasticity and cognitive function areimpaired in the hippocampus of old versus young animals. In FIG. 6Asynaptic plasticity of normal aging animals was examined in hippocampalslices by extracellular electrophysiological recordings using along-term potentiation (LTP) paradigm. LTP levels recorded from the DGwere lower in the hippocampus of old (100.25±14.0%, n=7) versus young(201.1±40.6%, n=6) animals following 40 minutes after induction. FIG. 6Bshows how spatial learning and memory was assessed during normal agingin young (2-3 months) versus old (18-20 months) adult animals (7-8C57BI/6 male mice per group). Old mice demonstrate impaired learning andmemory for platform location during the testing phase of the task.Cognitive deficits were quantified as the number of entry arm errorsmade prior to finding the target platform. All data is represented asMean±SEM; *, P<0.05; **, P<0.01; ANOVA, Tukey's post-hoc test.

FIGS. 7A-7F show that heterochronic parabiosis reduces proliferation andprogenitor frequency in the DG of young animals while increasingproliferation in aged animals. After five weeks of parabiosis, animalswere injected with BrdU for three days prior to sacrifice. BrdUimmunostaining was performed for young (3-4 months) and aged (18-20months) isochronic and heterochronic parabionts. FIG. 7A showsquantification of proliferation in the young DG after parabiosis. Dataare from 8 mice for isochronic and 6 mice for heterochronic groups. FIG.7B shows quantification of proliferation in the aged DG afterparabiosis. Data are from 4 mice for isochronic and 6 mice forheterochronic groups. Sox2 immunostaining was also performed for young(3-4 months) isochronic and heterochronic parabionts. FIG. 7C showsquantification of Sox2-positive progenitor cells in the young DG afterparabiosis. Data are from 8 mice for isochronic and 6 mice forheterochronic groups. FIGS. 7D and 7E show quantification ofneurogenesis (Dcx, Doublecortin-positive cells) in the DG during normalaging and after isochronic (Iso) or heterochronic (Het) parabiosis. 7Adata are from 10 normal aged (18 months old) mice, 6 isochronicparabionts (18-20 months old) and 12 heterochronic parabionts (18-20months old). 7F shows quantification of neurite length during normalaging and after parabiosis in Dcx-positive cells. Dendritic lengthremained unchanged between unpaired normal aged animals and isochronicparabiotic animals. All data are from 5-7 sections per mouse; bars aremean±SEM; * P<0.05; ** P<0.01; n.s., not significant; t-test.

FIGS. 8A-8E show that circulatory system is shared between animalsduring parabiosis. FIGS. 8A-8D show a subset of four parabiotic pairswere generated by joining young (2-3 months old) actin-GFP transgenicwith young (2-3 months old) and aged (18 months old) non-transgenicmice. Blood was isolated two weeks after surgery and flow cytometricanalysis was done on fixed and permeabilized blood cells. Representativeflow-cytometry plots demonstrate the frequency of GFP-positive cells ina GFP-transgenic (tg) parabiont (a,c) and wild-type (wt) parabiont (8B,8D) at the time of sacrifice. MFI, mean fluorescence intensity. FIG. 8Eshows quantification of GFP-positive cells in the DG of the hippocampusin young and aged wild-type parabionts after parabiosis with youngactin-GFP-positive parabionts. 5 sections per mouse; bars are mean±SEM;n.s., not significant; t-test.

FIGS. 9A-9C show that changes in concentrations of selected secretedplasma proteins correlate with declining neurogenesis in aging andheterochronic parabiosis. FIG. 9A shows an analysis of plasma proteincorrelations with decreased neurogenesis in the aging mouse samplesusing the Significance Analysis of Microarray software (SAM 3.00algorithm). SAM assigns d-scores to each gene or protein on the basis ofa multi-comparison analysis of expression changes and indicatessignificance by q-value. FIG. 9B shows unsupervised clustering ofsecreted signaling factors that were significantly associated withage-related decreased neurogenesis with a false discovery rate of 7.34%or less (SAM, q 7.34). Mouse age groups are indicated at the top of thenode map as boxes in which youngest ages are tan and oldest ages arered. Thus cluster analysis of systemic factors associated with decreasedneurogenesis also produce a reasonable separation of samples by age.Color shades in the node map indicate higher (purple) or lower (green)relative plasma concentrations. FIG. 9C shows quantitative fold changesin soluble signaling factors between isochronic versus heterochronicparabiotic groups. Color shades indicate increases (darker gray scale)and decreases (lighter grey scale) in relative plasma concentrations(mean±SEM of fold changes observed with parabiosis; n.c. denotes nosignificant change).

FIGS. 10A-C show that systemic administration of CCL11 reduces cellproliferation but not glial differentiation in the DG of young animals.Young adult male mice (2-3 months old) were injected with eitherrecombinant murine CCL11 or PBS (vehicle) through intraperitonealinjections every three days for ten days for a total of four injectionsAnimals were injected with BrdU for three days prior to sacrifice. FIG.10A shows that a significant increase above basal CCL11 plasma levelswas measured in mice treated systemically with recombinant CCL11, but norelative change was observed in animals receiving PBS. Blood wascollected by mandibular vein bleed prior to systemic drug administrationand by intracardial bleed at time of sacrifice using EDTA as ananticoagulant. Plasma was generated by centrifugation of blood. Sampleswere diluted 1:10 and CCL11 was detected by Quantikine ELISA followingthe manufacturers manual (R&D Systems). BrdU immunostaining wasperformed in the DG for each treatment group. FIG. 10B showsquantification of BrdU-positive cells in the DG after systemic drugadministration. Data are from 5-10 mice per group (5 sections permouse). Confocal microscopy images from the subgranular zone of the DGof brain sections immunostained for BrdU in combination with GFAP wasalso performed for both treatment groups. FIG. 10C shows quantificationof the relative number of BrdU and GFAP double positive cells out of allBrdU-positive cells in the DG after systemic CCL11 administration. Dataare from 5 mice per group (3 sections per mouse). Bars show mean±SEM; *,P<0.05; **, P<0.01; n.s., not significant; t-test (10C) or ANOVA,Dunnet's post-hoc test (10A, 10B).

FIGS. 11A-11C show that systemic administration of MCSF does not alterneurogenesis in the DG of young animals. FIGS. 11A and 11B show acomparison of plasma concentrations for MCSF in normal aged (6, 12, 18and 24 months old) (11A) and young heterochronic parabionts pre and postparabiotic pairing (11B). Young adult male mice (2-3 months old) wereinjected with either recombinant MCSF alone or PBS as a vehicle controlthrough intraperitoneal injections every three days for ten days.Neurogenesis was analyzed by immunostaining for Dcx. FIG. 11C showsquantification of neurogenesis in the DG after systemic drugadministration. Data are from 5 mice per group (5 sections per mouse).Bars show mean±SEM; n.s, not significant; t-test (11B and 11C) or ANOVA,Dunnet's post-hoc test (11A).

FIGS. 12A-12H show that age-related blood borne factors, including CCL11and CCL2, inhibit NPC function and neural differentiation in vitro. FIG.12A shows an experiment where primary NPCs were exposed to serumisolated from young (2-3 months) or old (18-22 months) mice for fourdays in culture under self-renewal conditions. The number ofneurospheres formed in the presence of old serum was decreased comparedto neurospheres formed in the presence of young serum. FIG. 12B shows adose-dependent decrease in the number of neurospheres formed fromprimary mouse NPCs after exposure to murine recombinant CCL11 for fourdays in culture under self-renewal conditions. FIG. 12C shows decreasein neurosphere formation after exposure to murine recombinant CCL11compared with PBS (vehicle) control is rescued by addition of anti-CCL11neutralizing antibody but not by a non-specific isotype controlantibody. FIG. 12D shows a decrease in the number of neurospheres formedfrom primary mouse NPCs after exposure to murine recombinant CCL2 isrescued by addition of anti-CCL2 neutralizing antibody. FIGS. 12E-F showdecreased neurosphere size and quantitation thereof after exposure toCCL11. FIG. 12G shows a quantification of decreased neuronaldifferentiation as a function of reduced expression of Dcxpromoter-controlled eGFP in stably transfected human derived NTERA cellsafter exposure to human recombinant CCL11 (12G) or CCL2 (12H), comparedwith PBS (vehicle) as a control. FIG. 12G shows that decreased neuronaldifferentiation is rescued by addition of anti-CCL11 neutralizingantibody but not by a non-specific isotype control antibody. FIG. 12Hshows quantification of dose dependent decrease in neuronaldifferentiation after exposure to human recombinant CCL2. HumanNTERA-EGFP reporter cells were cultured under differentiation conditions(RA, retinoic acid) for 12 days and relative Dxc reporter gene activitywas measured as fluorescence intensity. In vitro data are representativeof three independent experiments done in triplicate. Bars are mean±SEM;*, P<0.05; **, P<0.01; ***, P<0.001; t-test (a,f) or ANOVA, Dunnett'spost-hoc test (12B-12D, 12G, 12H).

FIG. 13 shows that neurogenesis is inhibited by direct exposure to CCL11in vivo. Young adult mice were injected stereotaxically with eitherrecombinant CCL11 or PBS into the left or right DG. Dcx-positive cellsin adjacent sides of the DG within the same section were shown fortreatment groups. Quantification of neurogenesis in the DG afterstereotactic CCL11 administration is shown. All data are from 4-5 youngadult mice (2-3 months of age) per group (5 sections per mouse). Barsshow mean±SEM; *, P<0.05; t-test

FIGS. 14A-14B show a proposed model illustrating the cellular andfunctional impact of age-related systemic molecular changes on the adultneurogenic niche. Schematic of cellular changes occurring in theneurogenic niche during normal aging and heterochronic parabiosis.Levels of blood-borne factors, including the chemokines CCL11 and CCL2,increase during normal aging and heterochronic parabiosis. Thesesystemic changes contribute to the decline in neurogenesis observed inthe adult brain and functionally impair synaptic plasticity and learningand memory. Cellular impact illustration is provided in FIG. 14A andfunctional impact scenario is provided in FIG. 14B. Cell typesillustrated include neural stem cells (NPC), neurons, astrocytes, andmicroglia (FIG. 14A).

DETAILED DESCRIPTION

Methods of treating an adult mammal for an aging-associated impairmentare provided. Aspects of the methods include modulating CCR3, e.g., viamodulating eotaxin-1/CCR3 interaction, in the mammal in a mannersufficient to treat the mammal for the aging-associated impairment. Avariety of aging-associated impairments may be treated by practice ofthe methods, which impairments include cognitive impairments.

Before the present methods and compositions are described, it is to beunderstood that this invention is not limited to a particular method orcomposition described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof,e.g., polypeptides, known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Methods

As summarized above, aspects of the invention include methods oftreating an aging-associated impairment in an adult mammal. Theaging-associated impairment may manifest in a number of different ways,e.g., as aging-associated cognitive impairment and/or physiologicalimpairment, e.g., in the form of damage to central or peripheral organsof the body, such as but not limited to: cell injury, tissue damage,organ dysfunction, aging-associated lifespan shortening andcarcinogenesis, where specific organs and tissues of interest include,but are not limited to skin, neuron, muscle, pancreas, brain, kidney,lung, stomach, intestine, spleen, heart, adipose tissue, testes, ovary,uterus, liver and bone; in the form of decreased neurogenesis, etc.

In some embodiments, the aging-associated impairment is anaging-associated impairment in cognitive ability in an individual, i.e.,an aging-associated cognitive impairment. By cognitive ability, or“cognition”, it is meant the mental processes that include attention andconcentration, learning complex tasks and concepts, memory (acquiring,retaining, and retrieving new information in the short and/or longterm), information processing (dealing with information gathered by thefive senses), visuospatial function (visual perception, depthperception, using mental imagery, copying drawings, constructing objectsor shapes), producing and understanding language, verbal fluency(word-finding), solving problems, making decisions, and executivefunctions (planning and prioritizing). By “cognitive decline”, it ismeant a progressive decrease in one or more of these abilities, e.g., adecline in memory, language, thinking, judgment, etc. By “an impairmentin cognitive ability” and “cognitive impairment”, it is meant areduction in cognitive ability relative to a healthy individual, e.g.,an age-matched healthy individual, or relative to the ability of theindividual at an earlier point in time, e.g., 2 weeks, 1 month, 2months, 3 months, 6 months, 1 year, 2 years, 5 years, or 10 years ormore previously. Aging-associated cognitive impairments includeimpairments in cognitive ability that are typically associated withaging, including, for example, cognitive impairment associated with thenatural aging process, e.g., mild cognitive impairment (M.C.I.); andcognitive impairment associated with an aging-associated disorder, thatis, a disorder that is seen with increasing frequency with increasingsenescence, e.g., a neurodegenerative condition such as Alzheimer'sdisease, Parkinson's disease, frontotemporal dementia, Huntington'sdisease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma,myotonic dystrophy, vascular dementia, and the like.

By “treatment” it is meant that at least an amelioration of one or moresymptoms associated with an aging-associated impairment afflicting theadult mammal is achieved, where amelioration is used in a broad sense torefer to at least a reduction in the magnitude of a parameter, e.g., asymptom associated with the impairment being treated. As such, treatmentalso includes situations where a pathological condition, or at leastsymptoms associated therewith, are completely inhibited, e.g., preventedfrom happening, or stopped, e.g., terminated, such that the adult mammalno longer suffers from the impairment, or at least the symptoms thatcharacterize the impairment. In some instances, “treatment”, “treating”and the like refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete cure for a diseaseand/or adverse effect attributable to the disease. “Treatment” may beany treatment of a disease in a mammal, and includes: (a) preventing thedisease from occurring in a subject which may be predisposed to thedisease but has not yet been diagnosed as having it; (b) inhibiting thedisease, i.e., arresting its development; or (c) relieving the disease,i.e., causing regression of the disease. Treatment may result in avariety of different physical manifestations, e.g., modulation in geneexpression, increased neurogenesis, rejuvenation of tissue or organs,etc. Treatment of ongoing disease, where the treatment stabilizes orreduces the undesirable clinical symptoms of the patient, occurs in someembodiments. Such treatment may be performed prior to complete loss offunction in the affected tissues. The subject therapy may beadministered during the symptomatic stage of the disease, and in somecases after the symptomatic stage of the disease.

In some instances where the aging-associated impairment isaging-associated cognitive decline, treatment by methods of the presentdisclosure slows, or reduces, the progression of aging-associatedcognitive decline. In other words, cognitive abilities in the individualdecline more slowly, if at all, following treatment by the disclosedmethods than prior to or in the absence of treatment by the disclosedmethods. In some instances, treatment by methods of the presentdisclosure stabilizes the cognitive abilities of an individual. Forexample, the progression of cognitive decline in an individual sufferingfrom aging-associated cognitive decline is halted following treatment bythe disclosed methods. As another example, cognitive decline in anindividual, e.g., an individual 40 years old or older, that is projectedto suffer from aging-associated cognitive decline, is preventedfollowing treatment by the disclosed methods. In other words, no(further) cognitive impairment is observed. In some instances, treatmentby methods of the present disclosure reduces, or reverses, cognitiveimpairment, e.g., as observed by improving cognitive abilities in anindividual suffering from aging-associated cognitive decline. In otherwords, the cognitive abilities of the individual suffering fromaging-associated cognitive decline following treatment by the disclosedmethods are better than they were prior to treatment by the disclosedmethods, i.e., they improve upon treatment. In some instances, treatmentby methods of the present disclosure abrogates cognitive impairment. Inother words, the cognitive abilities of the individual suffering fromaging-associated cognitive decline are restored, e.g., to their levelwhen the individual was about 40 years old or less, following treatmentby the disclosed methods, e.g., as evidenced by improved cognitiveabilities in an individual suffering from aging-associated cognitivedecline.

In some instances, treatment of an adult mammal in accordance with themethods results in a change in a central organ, e.g., a central nervoussystem organ, such as the brain, spinal cord, etc., where the change maymanifest in a number of different ways, e.g., as described in greaterdetail below, including but not limited to molecular, structural and/orfunctional, e.g., in the form of enhanced neurogenesis.

As summarized above, methods described herein are methods of treating anaging-associated impairment, e.g., as described above, in an adultmammal. By adult mammal is meant a mammal that has reached maturity,i.e., that is fully developed. As such, adult mammals are not juvenile.Mammalian species that may be treated with the present methods includecanines and felines; equines; bovines; ovines; etc., and primates,including humans. The subject methods, compositions, and reagents mayalso be applied to animal models, including small mammals, e.g., murine,lagomorpha, etc., for example, in experimental investigations. Thediscussion below will focus on the application of the subject methods,compositions, reagents, devices and kits to humans, but it will beunderstood by the ordinarily skilled artisan that such descriptions canbe readily modified to other mammals of interest based on the knowledgein the art.

The age of the adult mammal may vary, depending on the type of mammalthat is being treated. Where the adult mammal is a human, the age of thehuman is generally 18 years or older. In some instances, the adultmammal is an individual suffering from or at risk of suffering from anaging-associated impairment, such as an aging-associated cognitiveimpairment, where the adult mammal may be one that has been determined,e.g., in the form of receiving a diagnosis, to be suffering from or atrisk of suffering from an aging-associated impairment, such as anaging-associated cognitive impairment. The phrase “an individualsuffering from or at risk of suffering from an aging-associatedcognitive impairment” refers to an individual that is about 50 years oldor older, e.g., 60 years old or older, 70 years old or older, 80 yearsold or older, and sometimes no older than 100 years old, such as 90years old, i.e., between the ages of about 50 and 100, e.g., 50, 55, 60,65, 70, 75, 80, 85 or about 90 years old. The individual may suffer froman aging associated condition, e.g., cognitive impairment, associatedwith the natural aging process, e.g., M.C.I. Alternatively, theindividual may be 50 years old or older, e.g., 60 years old or older, 70years old or older, 80 years old or older, 90 years old or older, andsometimes no older than 100 years old, i.e., between the ages of about50 and 100, e.g., 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or about 100years old, and has not yet begun to show symptoms of an aging associatedcondition, e.g., cognitive impairment. In yet other embodiments, theindividual may be of any age where the individual is suffering from acognitive impairment due to an aging-associated disease, e.g.,Alzheimer's disease, Parkinson's disease, frontotemporal dementia,Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis,glaucoma, myotonic dystrophy, dementia, and the like. In some instances,the individual is an individual of any age that has been diagnosed withan aging-associated disease that is typically accompanied by cognitiveimpairment, e.g., Alzheimer's disease, Parkinson's disease,frontotemporal dementia, progressive supranuclear palsy, Huntington'sdisease, amyotrophic lateral sclerosis, spinal muscular atrophy,multiple sclerosis, multi-system atrophy, glaucoma, ataxias, myotonicdystrophy, dementia, and the like, where the individual has not yetbegun to show symptoms of cognitive impairment.

As summarized above, aspects of the methods include modulating CCR3. Bymodulating CCR3 is meant changing its activity in a manner sufficient totreat the mammal for the target aging-associated impairment. Modulatingmay be accomplished in a variety of different ways, e.g., by changingthe ability of CCR3 to interact with one or of its ligands, by changingthe expression level of CCR3, etc., as described in greater detailbelow. In some instances, modulating CCR3 including modulatingeotaxin-1/CCR3 interaction in the mammal in a manner sufficient to treatthe aging impairment in the mammal, e.g., as described above. Bymodulating eotaxin-1/CCR3 interaction is meant changing the interactionof eotaxin-1 (i.e., C—C motif chemokine 11, CCL11, eosinophilchemotactic protein) with CCR3 (i.e., C—C chemokine receptor type 3,CD193) in a manner sufficient to achieve the desired treatment. Theinteraction of eotaxin-1 with CCR3 may be changed using a variety ofdifferent approaches, e.g., as described below, including interferingwith binding of eotaxin-1 and CCR3, reducing the level of activeeotaxin-1 and/or CCR3, etc.

In some instances, the eotaxin-1/CCR3 interaction is modulated byreducing active systemic eotaxin-1 in the mammal. By reducing activesystemic eotaxin-1 is meant lowering the amount or level of activeeotaxin-1 that is systemically present in (i.e., in the circulatorysystem of) the mammal, such as the amount of active extracellulareotaxin-1 that is present in the cardiovascular system of the mammal.While the magnitude of the reduction may vary, in some instances themagnitude is 2-fold or greater, such as 5-fold or greater, including10-fold or greater, e.g., 15-fold or greater, 20-fold or greater,25-fold or greater (as compared to a suitable control), where in someinstances the magnitude is such that the amount of detectable active(e.g., free) eotaxin-1 in the circulatory system of the individual is50% or less, such as 25% or less, including 10% or less, e.g., 1% orless, relative to the amount that was detectable prior to interventionaccording to the invention, and in some instances the amount isundetectable following intervention.

The eotaxin-1 level may be reduced using any convenient protocol. Insome embodiments, the eotaxin-1 level is reduced by administering to themammal an effective amount of an active system eotaxin-1 reducing agent,i.e., an agent whose administration results in the reduction of activeeotaxin-1 (e.g., eotaxin-1 that can bind to CCR3) that is systemicallypresent in the mammal. As such, in practicing methods according to theseembodiments of the invention, an effective amount of the active agent,e.g., eotaxin-1 modulatory agent, is provided to the adult mammal.

Depending on the particular embodiments being practiced, a variety ofdifferent types of active agents may be employed. In some instances, theagent is an agent that modulates, e.g., inhibits, eotaxin-1 activity bybinding to eotaxin-1 and/or inhibiting binding of eotaxin-1 to areceptor therefore, e.g., CCR3. For example, agents that bind toeotaxin-1 and inhibit its activity are of interest. In certain of theseembodiments, the administered active agent is an eotaxin-1 specificbinding member. In general, useful eotaxin-1 specific binding membersexhibit an affinity (Kd) for a target eotaxin-1, such as humaneotaxin-1, that is sufficient to provide for the desired reduction inaging associated impairment eotaxin-1 activity. As used herein, the term“affinity” refers to the equilibrium constant for the reversible bindingof two agents; “affinity” can be expressed as a dissociation constant(Kd). Affinity can be at least 1-fold greater, at least 2-fold greater,at least 3-fold greater, at least 4-fold greater, at least 5-foldgreater, at least 6-fold greater, at least 7-fold greater, at least8-fold greater, at least 9-fold greater, at least 10-fold greater, atleast 20-fold greater, at least 30-fold greater, at least 40-foldgreater, at least 50-fold greater, at least 60-fold greater, at least70-fold greater, at least 80-fold greater, at least 90-fold greater, atleast 100-fold greater, or at least 1000-fold greater, or more, than theaffinity of an antibody for unrelated amino acid sequences. Affinity ofa specific binding member to a target protein can be, for example, fromabout 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or more.The term “binding” refers to a direct association between two molecules,due to, for example, covalent, electrostatic, hydrophobic, and ionicand/or hydrogen-bond interactions, including interactions such as saltbridges and water bridges. In some embodiments, the antibodies bindhuman eotaxin-1 with nanomolar affinity or picomolar affinity. In someembodiments, the antibodies bind human eotaxin-1 with a Kd of less thanabout 100 nM, 50 nM, 20 nM, 20 nM, or 1 nM. In some embodiments, theaffinity between the binding member active agent in a binding complexwith eotaxin-1 is characterized by a K_(d) (dissociation constant) of10⁻⁶ M or less, such as 10⁻⁷ M or less, including 10⁻⁸ M or less, e.g.,10⁻⁹ M or less, 10⁻¹⁰ M or less, 10⁻¹¹ M or less, 10⁻¹² M or less, 10⁻¹³M or less, 10⁻¹⁴ M or less, including 10⁻¹⁵ M or less.

Examples of eotaxin-1 specific binding members include eotaxin-1antibodies and binding fragments thereof. Non-limiting examples of suchantibodies include antibodies directed against any epitope of eotaxin-1.Also encompassed are bispecific antibodies, i.e., antibodies in whicheach of the two binding domains recognizes a different binding epitope.Cloning of human eotaxin-1 was reported in Ponath et al., “Cloning ofthe human eosinophil chemoattractant, eotaxin: expression, receptorbinding, and functional properties suggest a mechanism for the selectiverecruitment of eosinophils,” J. Clin. Invest. (1996) 97: 604-612. Theamino acid sequence of human eotaxin-1 is MKVSAALLWL LLIAAAFSPQGLAGPASVPT TCCFNLANRK IPLQRLESYR RITSGKCPQK AVIFKTKLAK DICADPKKKWVQDSMKYLDQ KSPTPKP (SEQ ID NO:01).

Antibody specific binding members that may be employed include fullantibodies or immunoglobulins of any isotype, as well as fragments ofantibodies which retain specific binding to antigen, including, but notlimited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies,humanized antibodies, single-chain antibodies, and fusion proteinscomprising an antigen-binding portion of an antibody and a non-antibodyprotein. The antibodies may be detectably labeled, e.g., with aradioisotope, an enzyme which generates a detectable product, afluorescent protein, and the like. The antibodies may be furtherconjugated to other moieties, such as members of specific binding pairs,e.g., biotin (member of biotin-avidin specific binding pair), and thelike. Also encompassed by the term are Fab′, Fv, F(ab′)2, and or otherantibody fragments that retain specific binding to antigen, andmonoclonal antibodies. An antibody may be monovalent or bivalent.

“Antibody fragments” comprise a portion of an intact antibody, forexample, the antigen binding or variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fvfragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10): 1057-1062 (1995)); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments. Papaindigestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two antigen combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRS of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The “Fab” fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxyl terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)2 antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains. Depending on the amino acid sequence of the constant domain oftheir heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

“Single-chain Fv” or “sFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. In some embodiments, the Fv polypeptide furthercomprises a polypeptide linker between the VH and VL domains, whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

Antibodies that may be used in connection with the present disclosurethus can encompass monoclonal antibodies, polyclonal antibodies,bispecific antibodies, Fab antibody fragments, F(ab)2 antibodyfragments, Fv antibody fragments (e.g., VH or VL), single chain Fvantibody fragments and dsFv antibody fragments. Furthermore, theantibody molecules may be fully human antibodies, humanized antibodies,or chimeric antibodies. In some embodiments, the antibody molecules aremonoclonal, fully human antibodies.

The antibodies that may be used in connection with the presentdisclosure can include any antibody variable region, mature orunprocessed, linked to any immunoglobulin constant region. If a lightchain variable region is linked to a constant region, it can be a kappachain constant region. If a heavy chain variable region is linked to aconstant region, it can be a human gamma 1, gamma 2, gamma 3 or gamma 4constant region, more preferably, gamma 1, gamma 2 or gamma 4 and evenmore preferably gamma 1 or gamma 4.

In some embodiments, fully human monoclonal antibodies directed againsteotaxin are generated using transgenic mice carrying parts of the humanimmune system rather than the mouse system.

Minor variations in the amino acid sequences of antibodies orimmunoglobulin molecules are encompassed by the present invention,providing that the variations in the amino acid sequence maintain atleast 75%, e.g., at least 80%, 90%, 95%, or 99% of the sequence. Inparticular, conservative amino acid replacements are contemplated.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Whether an amino acidchange results in a functional peptide can readily be determined byassaying the specific activity of the polypeptide derivative. Fragments(or analogs) of antibodies or immunoglobulin molecules, can be readilyprepared by those of ordinary skill in the art. Preferred amino- andcarboxy-termini of fragments or analogs occur near boundaries offunctional domains. Structural and functional domains can be identifiedby comparison of the nucleotide and/or amino acid sequence data topublic or proprietary sequence databases. Preferably, computerizedcomparison methods are used to identify sequence motifs or predictedprotein conformation domains that occur in other proteins of knownstructure and/or function. Methods to identify protein sequences thatfold into a known three-dimensional structure are known. Sequence motifsand structural conformations may be used to define structural andfunctional domains in accordance with the invention.

Specific examples of antibody agents that may be employed to reduce thelevel of active systemic eotaxin-1 include, but are not limited to:bertilimumab (i.e., iCo-008 or CAT-213) as further described in Main etal., “A Potent Human Anti-Eotaxin1 Antibody, CAT-213: Isolation by PhageDisplay and in Vitro and in Vivo Efficacy,” JPET (2006) 319: 1395-1404;MAB320, AF-320-NA and MAB3201 from R & D Systems; ANT-126 available fromProspec; as well as the antibodies described in U.S. Pat. Nos. 6,946,546and 7,323,311; the disclosures of which are herein incorporated byreference.

Eotaxin-1 binding agents that may be employed also include smallmolecules that bind to the eotaxin-1 and inhibit its activity, i.e.,small molecule eotaxin-1 antagonists. Naturally occurring or syntheticsmall molecule compounds of interest include numerous chemical classes,such as organic molecules, e.g., small organic compounds having amolecular weight of more than 50 and less than about 2,500 daltons.Candidate agents comprise functional groups for structural interactionwith proteins, particularly hydrogen bonding, and typically include atleast an amine, carbonyl, hydroxyl or carboxyl group, preferably atleast two of the functional chemical groups. The candidate agents mayinclude cyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomoleculesincluding peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.Such molecules may be identified, among other ways, by employing thescreening protocols described below.

In some instances, the agent modulates expression of the RNA and/orprotein from the gene, such that it changes the expression of the RNA orprotein from the target gene in some manner. In these instances, theagent may change expression of the RNA or protein in a number ofdifferent ways. In certain embodiments, the agent is one that reduces,including inhibits, expression of an eotaxin-1 protein. Inhibition ofeotaxin-1 protein expression may be accomplished using any convenientmeans, including use of an agent that inhibits eotaxin-1 proteinexpression, such as, but not limited to: RNAi agents, antisense agents,agents that interfere with a transcription factor binding to a promotersequence of the eotaxin-1 gene, or inactivation of the eotaxin-1 gene,e.g., through recombinant techniques, etc.

For example, the transcription level of an eotaxin-1 protein can beregulated by gene silencing using RNAi agents, e.g., double-strand RNA(see e.g., Sharp, Genes and Development (1999) 13: 139-141). RNAi, suchas double-stranded RNA interference (dsRNAi) or small interfering RNA(siRNA), has been extensively documented in the nematode C. elegans(Fire, et al, Nature (1998) 391:806-811) and routinely used to “knockdown” genes in various systems. RNAi agents may be dsRNA or atranscriptional template of the interfering ribonucleic acid which canbe used to produce dsRNA in a cell. In these embodiments, thetranscriptional template may be a DNA that encodes the interferingribonucleic acid. Methods and procedures associated with RNAi are alsodescribed in published PCT Application Publication Nos. WO 03/010180 andWO 01/68836, the disclosures of which applications are incorporatedherein by reference. dsRNA can be prepared according to any of a numberof methods that are known in the art, including in vitro and in vivomethods, as well as by synthetic chemistry approaches. Examples of suchmethods include, but are not limited to, the methods described by Sadheret al., Biochem. Int. (1987) 14:1015; Bhattacharyya, Nature (1990)343:484; and U.S. Pat. No. 5,795,715, the disclosures of which areincorporated herein by reference. Single-stranded RNA can also beproduced using a combination of enzymatic and organic synthesis or bytotal organic synthesis. The use of synthetic chemical methods enableone to introduce desired modified nucleotides or nucleotide analogs intothe dsRNA. dsRNA can also be prepared in vivo according to a number ofestablished methods (see, e.g., Sambrook, et al. (1989) MolecularCloning: A Laboratory Manual, 2nd ed.; Transcription and Translation (B.D. Hames, and S. J. Higgins, Eds., 1984); DNA Cloning, volumes I and II(D. N. Glover, Ed., 1985); and Oligonucleotide Synthesis (M. J. Gait,Ed., 1984, each of which is incorporated herein by reference). A numberof options can be utilized to deliver the dsRNA into a cell orpopulation of cells such as in a cell culture, tissue, organ or embryo.For instance, RNA can be directly introduced intracellularly. Variousphysical methods are generally utilized in such instances, such asadministration by microinjection (see, e.g., Zernicka-Goetz, et al.Development (1997)124:1133-1137; and Wianny, et al., Chromosoma (1998)107: 430-439). Other options for cellular delivery includepermeabilizing the cell membrane and electroporation in the presence ofthe dsRNA, liposome-mediated transfection, or transfection usingchemicals such as calcium phosphate. A number of established genetherapy techniques can also be utilized to introduce the dsRNA into acell. By introducing a viral construct within a viral particle, forinstance, one can achieve efficient introduction of an expressionconstruct into the cell and transcription of the RNA encoded by theconstruct. Specific examples of RNAi agents that may be employed toreduce eotaxin-1 expression include, but are not limited to: MBS8238622from MyBioSource; CCL11 (Gene ID 6356) Human shRNA available fromOriGene (Référence SR304280); CCL11 siRNA/shRNA/RNAi Lentivirus (Human)(Target a) available from ABM; etc.

In some instances, antisense molecules can be used to down-regulateexpression of an eotaxin-1 gene in the cell. The anti-sense reagent maybe antisense oligodeoxynucleotides (ODN), particularly synthetic ODNhaving chemical modifications from native nucleic acids, or nucleic acidconstructs that express such anti-sense molecules as RNA. The antisensesequence is complementary to the mRNA of the targeted protein, andinhibits expression of the targeted protein. Antisense molecules inhibitgene expression through various mechanisms, e.g., by reducing the amountof mRNA available for translation, through activation of RNAse H, orsteric hindrance. One or a combination of antisense molecules may beadministered, where a combination may include multiple differentsequences.

Antisense molecules may be produced by expression of all or a part ofthe target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. Shortoligonucleotides, of from 7 to 8 bases in length, can be strong andselective inhibitors of gene expression (see Wagner et al., NatureBiotechnol. (1996)14:840-844).

A specific region or regions of the endogenous sense strand mRNAsequence are chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in an in vitro or animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

Antisense oligonucleotides may be chemically synthesized by methodsknown in the art (see Wagner et al. (1993), supra.) Oligonucleotides maybe chemically modified from the native phosphodiester structure, inorder to increase their intracellular stability and binding affinity. Anumber of such modifications have been described in the literature,which alter the chemistry of the backbone, sugars or heterocyclic bases.Among useful changes in the backbone chemistry are phosphorothioates;phosphorodithioates, where both of the non-bridging oxygens aresubstituted with sulfur; phosphoroamidites; alkyl phosphotriesters andboranophosphates. Achiral phosphate derivatives include3′-O-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH₂-5′-O-phosphonate and 3′-NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity. The α-anomer of deoxyribose may be used, where the base isinverted with respect to the natural β-anomer. The 2′-OH of the ribosesugar may be altered to form 2′-O-methyl or 2′-O-allyl sugars, whichprovides resistance to degradation without comprising affinity.Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5-propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

As an alternative to anti-sense inhibitors, catalytic nucleic acidcompounds, e.g., ribozymes, anti-sense conjugates, etc., may be used toinhibit gene expression. Ribozymes may be synthesized in vitro andadministered to the patient, or may be encoded on an expression vector,from which the ribozyme is synthesized in the targeted cell (forexample, see International patent application WO 9523225, and Beigelmanet al., Nucl. Acids Res. (1995) 23:4434-42). Examples ofoligonucleotides with catalytic activity are described in WO 9506764.Conjugates of anti-sense ODN with a metal complex, e.g.terpyridylCu(II), capable of mediating mRNA hydrolysis are described inBashkin et al. Appl. Biochem. Biotechnol. (1995) 54:43-56.

In another embodiment, the eotaxin-1 gene is inactivated so that it nolonger expresses a functional protein. By inactivated is meant that thegene, e.g., coding sequence and/or regulatory elements thereof, isgenetically modified so that it no longer expresses a functionaleotaxin-1 protein, e.g., at least with respect to eotaxin-1 agingimpairment activity. The alteration or mutation may take a number ofdifferent forms, e.g., through deletion of one or more nucleotideresidues, through exchange of one or more nucleotide residues, and thelike. One means of making such alterations in the coding sequence is byhomologous recombination. Methods for generating targeted genemodifications through homologous recombination are known in the art,including those described in: U.S. Pat. Nos. 6,074,853; 5,998,209;5,998,144; 5,948,653; 5,925,544; 5,830,698; 5,780,296; 5,776,744;5,721,367; 5,614,396; 5,612,205; the disclosures of which are hereinincorporated by reference. Also of interest are CRISPR-CAS mediated genesilencing methods, e.g., as described in Published PCT Application Nos.WO/2015/071474; WO/2014/165825; WO/2015/006498; WO/2014/093595;WO/2015/089427; WO/2014/093694; WO/2015/021426; WO/2015/065964;WO/2015/089462; WO/2015/089486; WO/2014/093661; WO/2015/089419; thedisclosures of which are herein incorporated by reference.

Also of interest in certain embodiments are dominant negative mutants ofeotaxin-1 proteins, where expression of such mutants in the cell resultin a modulation, e.g., decrease, in eotaxin-1 mediated aging impairment.Dominant negative mutants of eotaxin-1 are mutant proteins that exhibitdominant negative eotaxin-1 activity. As used herein, the term“dominant-negative eotaxin-1 activity” or “dominant negative activity”refers to the inhibition, negation, or diminution of certain particularactivities of eotaxin-1, and specifically to eotaxin-1 mediated agingimpairment. Dominant negative mutations are readily generated forcorresponding proteins. These may act by several different mechanisms,including mutations in a substrate-binding domain; mutations in acatalytic domain; mutations in a protein binding domain (e.g., multimerforming, effector, or activating protein binding domains); mutations incellular localization domain, etc. A mutant polypeptide may interactwith wild-type polypeptides (made from the other allele) and form anon-functional multimer. In certain embodiments, the mutant polypeptidewill be overproduced. Point mutations are made that have such an effect.In addition, fusion of different polypeptides of various lengths to theterminus of a protein, or deletion of specific domains can yielddominant negative mutants. General strategies are available for makingdominant negative mutants (see for example, Herskowitz, Nature (1987)329:219, and the references cited above). Such techniques are used tocreate loss of function mutations, which are useful for determiningprotein function. Methods that are well known to those skilled in theart can be used to construct expression vectors containing codingsequences and appropriate transcriptional and translational controlsignals for increased expression of an exogenous gene introduced into acell. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.Alternatively, RNA capable of encoding gene product sequences may bechemically synthesized using, for example, synthesizers. See, forexample, the techniques described in “Oligonucleotide Synthesis”, 1984,Gait, M. J. ed., IRL Press, Oxford.

In some instances, the systemic acid level of eotaxin-1 is reduced byremoving systemic eotaxin-1 from the adult mammal, e.g., by removingeotaxin-1 from the circulatory system of the adult mammal. In suchinstances, any convenient protocol for removing circulatory eotaxin-1may be employed. For example, blood may be obtained from the adultmammal and extra-corporeally processed to remove eotaxin-1 from theblood to produce eotaxin-1 depleted blood, which resultant eotaxin-1depleted blood may then be returned to the adult mammal. Such protocolsmay employ a variety of different techniques in order to removeeotaxin-1 from the obtained blood. For example, the obtained blood maybe contacted with a filtering component, e.g., a membrane, etc., whichallows passage of eotaxin-1 but inhibits passage of other bloodcomponents, e.g., cells, etc. In some instances, the obtained blood maybe contacted with an eotaxin-1 absorptive component, e.g., porous beador particulate composition, which absorbs eotaxin-1 from the blood. Inyet other instances, the obtained blood may be contacted with aneotaxin-1 binding member stably associated with a solid support, suchthat eotaxin-1 binds to the binding member and is thereby immobilized onthe solid support, thereby providing for separation of eotaxin-1 fromother blood constituents. The protocol employed may or may not beconfigured to selectively remove eotaxin-1 from the obtained blood, asdesired.

In some instances, the eotaxin-1/CCR3 interaction is modulated byreducing active cell surface CCR3 in the mammal. By reducing active cellsurface CCR3 is meant lowering the amount or level of CCR3 that ispresent on cell surfaces and available for binding to eotaxin-1 in amanner that CCR3 is responsive to the presence of eotaxin-1. While themagnitude of the reduction may vary, in some instances the magnitude is2-fold or greater, such as 5-fold or greater, including 10-fold orgreater, e.g., 15-fold or greater, 20-fold or greater, 25-fold orgreater (as compared to a suitable control), where in some instances themagnitude is such that the amount of detectable cell surface active CCR3of the individual is 50% or less, such as 25% or less, including 10% orless, e.g., 1% or less, relative to the amount that was detectable priorto intervention according to the invention, and in some instances theamount is undetectable following intervention.

The active cell surface CCR3 level may be reduced using any convenientprotocol. In some embodiments, the active cell surface CCR3 level isreduced by administering to the mammal an effective amount of an activecell surface CCR3 reducing agent, i.e., an agent whose administrationresults in the reduction of cell surface active CCR3, e.g., CCR3 thatcan bind to eotaxin-1. As such, in practicing methods according to theseembodiments of the invention, an effective amount of the active agent,e.g., CCR3 modulatory agent, is provided to the adult mammal.

Depending on the particular embodiments being practiced, a variety ofdifferent types of active agents may be employed. In some instances, theagent is an agent that modulates, e.g., inhibits, CCR3 activity bybinding to CCR3 and/or inhibiting binding of CCR3 to a ligand therefore,e.g., eotaxin-1. For example, agents that bind to CCR3 and inhibit itsactivity are of interest. In certain of these embodiments, theadministered active agent is a CCR3 specific binding member. In general,useful CCR3 specific binding members exhibit an affinity (Kd) for atarget CCR3, such as human CCR3, that is sufficient to provide for thedesired reduction in aging associated impairment CCR3 activity. The term“affinity” and “binding” have the meanings provided above.

Examples of CCR3 specific binding members include CCR3 antibodies andbinding fragments thereof. Non-limiting examples of such antibodiesinclude antibodies directed against any epitope of CCR3, e.g., thesurface displayed epitope(s) of CCR3. Also encompassed are bispecificantibodies, i.e., antibodies in which each of the two binding domainsrecognizes a different binding epitope. Cloning of human CCR3 wasreported in Daugherty et al., “Cloning, expression, and characterizationof the human eosinophil eotaxin receptor,” J. Exp. Med. (1996) 183:2349-2354. The amino acid sequence of human CCR3 is MTTSLDTVETFGTTSYYDDV GLLCEKADTR ALMAQFVPPL YSLVFTVGLL GNVVVVMILI KYRRLRIMTNIYLLNLAISD LLFLVTLPFW IHYVRGHNWV FGHGMCKLLS GFYHTGLYSE IFFIILLTIDRYLAIVHAVF ALRARTVTFG VITSIVTWGL AVLAALPEFI FYETEELFEE TLCSALYPEDTVYSWRHFHT LRMTIFCLVL PLLVMAICYT GIIKTLLRCP SKKKYKAIRL IFVIMAVFFIFWTPYNVAIL LSSYQSILFG NDCERSKHLD LVMLVTEVIA YSHCCMNPVI YAFVGERFRKYLRHFFHRHL LMHLGRYIPF LPSEKLERTS SVSPSTAEPE LSIVF (SEQ ID NO:02).Antibody specific binding members that may be employed include fullantibodies or immunoglobulins of any isotype, as well as fragments ofantibodies which retain specific binding to antigen, including, but notlimited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies,humanized antibodies, single-chain antibodies, and fusion proteinscomprising an antigen-binding portion of an antibody and a non-antibodyprotein, e.g., as described above. Specific examples of CCR3 antibodiesinclude, but are not limited to: 12D5 monoclonal antibody (mAb), e.g.,as described in Li et al., Acta Trop. (2012) 121:118-24; MaB155 from R&D Systems; as well as those described in U.S. Pat. Nos. 6,207,155;6,610,834; 8,778,616

CCR3 binding agents that may be employed also include small moleculesthat bind to the CCR3 and inhibit its activity, i.e., CCR3 smallmolecule antagonists. Naturally occurring or synthetic small moleculecompounds of interest include numerous chemical classes, such as organicmolecules, e.g., small organic compounds having a molecular weight ofmore than 50 and less than about 2,500 daltons. Candidate agentscomprise functional groups for structural interaction with proteins,particularly hydrogen bonding, and typically include at least an amine,carbonyl, hydroxyl or carboxyl group, preferably at least two of thefunctional chemical groups. The candidate agents may include cyclicalcarbon or heterocyclic structures and/or aromatic or polyaromaticstructures substituted with one or more of the above functional groups.Candidate agents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof. Such molecules may beidentified, among other ways, by employing the screening protocolsdescribed below.

Specific examples of small molecule agents that bind to CCR3 include,but are not limited to: SB328437 (i.e.,(S)-Methyl-2-naphthoylamino-3-(4-nitrophenyl)propionate) which iscommercially available, for example from Calbiochem; SB 297006 (i.e.,N-Benzoyl-4-nitroaniline ethyl ester; W-56750 (i.e.,4-(3-aminophenyl)thiazol-2-ylthio]-N-[1-(3,4-dichlorobenzyl)piperidin-4-yl]acetamide) which is available commercially from Mitsubishi Tanabe PharmaCo.); GW766944 and GW824575 (GlaxoSmithKline); UCB 35625 (i.e.,1,4-trans-1-(1-Cycloocten-1-ylmethyl)-4-[[(2,7-dichloro-9H-xanthen-9-yl)carbonyl]amino]-1-ethylpiperidiniumiodide); piperidine derivatives, piperidine amides and biperidinecompounds such as those described in U.S. Pat. Nos. 6,984,651 and6,903,115, and U.S. published applications 20050176708, 20050182094 and20050182095 issued as 7,705,153; heterocyclic piperidines such as thosedescribed in U.S. Pat. No. 6,759,411; diphenyl-piperidine derivativessuch as those described in U.S. Pat. No. 6,566,376; 2,5-substitutedpyrimidine derivatives such as those described in U.S. Pat. No.6,984,643; piperizinones such as those described in U.S. Pat. No.6,974,869; cyclic amines such as those described in U.S. Pat. No.7,576,117; bicycylic and tricyclic amines such as those described inU.S. Pat. No. 6,960,666; N-ureidoalkyl-piperidines such as thosedescribed in U.S. Pat. Nos. 6,949,546, 6,919,368, 6,906,066, 6,897,234,6,875,776, 6,780,857, 6,627,629, 6,521,592 and 6,331,541; bicyclicdiamines such as those described in U.S. Pat. No. 6,821,964;benzylcycloalkyl amines such as those described in U.S. Pat. No.6,864,380; 2-substituted-4-nitrogen heterocycles such as those describedin U.S. Pat. No. 6,706,735; ureido derivatives ofpoly-4-amino-2-carboxy-I-methyl pyrrole compounds; bicyclic and bridgednitrogen heterocycles such as those described in U.S. publishedapplication 20050234034; azetidine derivatives such as those describedin U.S. published application 20050222118; substituted fused bicyclicamines such as those described in U.S. published application20050197373; substituted spiro azabicyclics such as those described inU.S. published application 20050197325; piperidine-substituted indolesor heteroderivatives thereof such as those described in U.S. publishedapplication 20050153979; piperidinyl and piperazinyl compoundssubstituted with bicyclo-heterocyclylalkyl groups such as thosedescribed in U.S. published application20050090504; arylsulfonamidederivatives such as those described in U.S. published application20050070582; 1-phenyl-1,2-diaminoethane derivatives such as thosedescribed in U.S. published application 20040063779;(N-{[2S]-4-(3,4-dichlorobenzyl)morpholin-2-yl}methyl)-N′[(2-methyl-2H-tetraazol-5-yl)methyl]urea)(see, e.g., Nakamura et al., Immunol Res., 33:213-222, 2006; the CCR3antagonist compounds described in Suzuki et al., Biochem. Biophys. Res.Commun., 339:1217-1223, 2006; Morokata et al., J. Pharmacol. Exp. Ther.,Dec. 9, 2005 [Epub ahead of print]); bipiperidine amide antagonists ofCCR3 such as those described in Ting et al., Bioorg. Med. Chem. Lett,15:3020-3023, 2005;(S)-methyl-2-naphthoylamino-3-(4-nitrophenyl)propionate (see, e.g.,Beasley et al., J. Allergy Clin. Immunol., 105: S466-S472, 2000; and theCCR3 antagonist compounds described in Fryer et al., J. Clin. Invest., 116:228-236, 2006; as well as those described in Kriegl et al.,Bioorganic & Medicinal Chemistry Letters (2015) 25: 229-235. In someinstances, the small molecule is not a compound described in U.S. Pat.No. 7,576,117 or 7,705,153.

In some instances, the agent modulates expression of the RNA and/orprotein from the gene, such that it changes the expression of the RNA orprotein from the target gene in some manner. In these instances, theagent may change expression of the RNA or protein in a number ofdifferent ways. In certain embodiments, the agent is one that reduces,including inhibits, expression of a CCR3 protein. Inhibition of CCR3protein expression may be accomplished using any convenient means,including use of an agent that inhibits eotaxin-1 protein expression,such as, but not limited to: RNAi agents, antisense agents, agents thatinterfere with a transcription factor binding to a promoter sequence ofthe CCR3 gene, or inactivation of the CCR3 gene, e.g., throughrecombinant techniques, etc. Further details regarding these varioustypes of active agents are provided above.

Also of interest in certain embodiments are dominant negative mutants ofCCR proteins, where expression of such mutants in the cell result in amodulation, e.g., decrease, in CCR3 mediated aging impairment. Detailsregarding dominant negative mutants are further described above.

In those embodiments where an active agent is administered to the adultmammal, the active agent(s) may be administered to the adult mammalusing any convenient administration protocol capable of resulting in thedesired activity. Thus, the agent can be incorporated into a variety offormulations, e.g., pharmaceutically acceptable vehicles, fortherapeutic administration. More particularly, the agents of the presentinvention can be formulated into pharmaceutical compositions bycombination with appropriate, pharmaceutically acceptable carriers ordiluents, and may be formulated into preparations in solid, semi-solid,liquid or gaseous forms, such as tablets, capsules, powders, granules,ointments (e.g., skin creams), solutions, suppositories, injections,inhalants and aerosols. As such, administration of the agents can beachieved in various ways, including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, transdermal, intracheal, etc.,administration.

In pharmaceutical dosage forms, the agents may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The agents can be utilized in aerosol formulation to be administered viainhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Where the agent is a polypeptide, polynucleotide, analog or mimeticthereof, it may be introduced into tissues or host cells by any numberof routes, including viral infection, microinjection, or fusion ofvesicles. Jet injection may also be used for intramuscularadministration, as described by Furth et al., Anal Biochem. (1992)205:365-368. The DNA may be coated onto gold microparticles, anddelivered intradermally by a particle bombardment device, or “gene gun”as described in the literature (see, for example, Tang et al., Nature(1992) 356:152-154), where gold microprojectiles are coated with theDNA, then bombarded into skin cells. For nucleic acid therapeuticagents, a number of different delivery vehicles find use, includingviral and non-viral vector systems, as are known in the art.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the nature of the deliveryvehicle, and the like. Preferred dosages for a given compound arereadily determinable by those of skill in the art by a variety of means.

In those embodiments where an effective amount of an active agent isadministered to the adult mammal, the amount or dosage is effective whenadministered for a suitable period of time, such as one week or longer,including two weeks or longer, such as 3 weeks or longer, 4 weeks orlonger, 8 weeks or longer, etc., so as to evidence a reduction in theimpairment, e.g., cognition decline and/or cognitive improvement in theadult mammal. For example, an effective dose is the dose that, whenadministered for a suitable period of time, such as at least about oneweek, and may be about two weeks, or more, up to a period of about 3weeks, 4 weeks, 8 weeks, or longer, will slow e.g., by about 20% ormore, e.g., by 30% or more, by 40% or more, or by 50% or more, in someinstances by 60% or more, by 70% or more, by 80% or more, or by 90% ormore, e.g., will halt, cognitive decline in a patient suffering fromnatural aging or an aging-associated disorder. In some instances, aneffective amount or dose of active agent will not only slow or halt theprogression of the disease condition but will also induce the reversalof the condition, i.e., will cause an improvement in cognitive ability.For example, in some instances, an effective amount is the amount thatwhen administered for a suitable period of time, usually at least aboutone week, and may be about two weeks, or more, up to a period of about 3weeks, 4 weeks, 8 weeks, or longer will improve the cognitive abilitiesof an individual suffering from an aging-associated cognitive impairmentby, for example 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, in someinstances 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more relative tocognition prior to administration of the blood product.

Where desired, effectiveness of treatment may be assessed using anyconvenient protocol. Cognition tests and IQ test for measuring cognitiveability, e.g., attention and concentration, the ability to learn complextasks and concepts, memory, information processing, visuospatialfunction, the ability to produce and understanding language, the abilityto solve problems and make decisions, and the ability to performexecutive functions, are well known in the art, any of which may be usedto measure the cognitive ability of the individual before and/or duringand after treatment with the subject blood product, e.g., to confirmthat an effective amount has been administered. These include, forexample, the General Practitioner Assessment of Cognition (GPCOG) test,the Memory Impairment Screen, the Mini Mental State Examination (MMSE),the California Verbal Learning Test, Second Edition, Short Form, formemory, the Delis-Kaplan Executive Functioning System test, theAlzheimer's Disease Assessment Scale (ADAS-Cog), the PsychogeriatricAssessment Scale (PAS) and the like. Progression of functional brainimprovements may be detected by brain imaging techniques, such asMagnetic Resonance Imaging (MRI) or Positron Emission Tomography (PET)and the like. A wide range of additional functional assessments may beapplied to monitor activities of daily living, executive functions,mobility, etc. In some embodiments, the method comprises the step ofmeasuring cognitive ability, and detecting a decreased rate of cognitivedecline, a stabilization of cognitive ability, and/or an increase incognitive ability after administration of the blood product as comparedto the cognitive ability of the individual before the blood product wasadministered. Such measurements may be made a week or more afteradministration of the blood product, e.g., 1 week, 2 weeks, 3 weeks, ormore, for instance, 4 weeks, 6 weeks, or 8 weeks or more, e.g., 3months, 4 months, 5 months, or 6 months or more.

Biochemically, by an “effective amount” or “effective dose” of activeagent is meant an amount of active agent that will inhibit, antagonize,decrease, reduce, or suppress by about 20% or more, e.g., by 30% ormore, by 40% or more, or by 50% or more, in some instances by 60% ormore, by 70% or more, by 80% or more, or by 90% or more, in some casesby about 100%, i.e., to negligible amounts, and in some instancesreverse, the reduction in synaptic plasticity and loss of synapses thatoccurs during the natural aging process or during the progression of anaging-associated disorder. In other words, cells present in adultmammals treated in accordance with methods of the invention will becomemore responsive to cues, e.g., activity cues, which promote theformation and maintenance of synapses.

Performance of methods of the invention, e.g., as described above, maymanifest as improvements in observed synaptic plasticity, both in vitroand in vivo as an induction of long term potentiation. For example, theinduction of LTP in neural circuits may be observed in awakeindividuals, e.g., by performing non-invasive stimulation techniques onawake individuals to induce LTP-like long-lasting changes in localizedneural activity (Cooke S F, Bliss T V (2006) Plasticity in the humancentral nervous system. Brain. 129(Pt 7):1659-73); mapping plasticityand increased neural circuit activity in individuals, e.g., by usingpositron emission tomography, functional magnetic resonance imaging,and/or transcranial magnetic stimulation (Cramer and Bastings, “Mappingclinically relevant plasticity after stroke,” Neuropharmacology(2000)39:842-51); and by detecting neural plasticity following learning,i.e., improvements in memory, e.g., by assaying retrieval-related brainactivity (Buchmann et al., “Prion protein M129V polymorphism affectsretrieval-related brain activity,” Neuropsychologia. (2008) 46:2389-402)or, e.g., by imaging brain tissue by functional magnetic resonanceimaging (fMRI) following repetition priming with familiar and unfamiliarobjects (Soldan et al., “Global familiarity of visual stimuli affectsrepetition-related neural plasticity but not repetition priming,”Neuroimage. (2008) 39:515-26; Soldan et al., “Aging does not affectbrain patterns of repetition effects associated with perceptual primingof novel objects,” J. Cogn. Neurosci. (2008) 20:1762-76). In someembodiments, the method includes the step of measuring synapticplasticity, and detecting a decreased rate of loss of synapticplasticity, a stabilization of synaptic plasticity, and/or an increasein synaptic plasticity after administration of the blood product ascompared to the synaptic plasticity of the individual before the bloodproduct was administered. Such measurements may be made a week or moreafter administration of the blood product, e.g., 1 week, 2 weeks, 3weeks, or more, for instance, 4 weeks, 6 weeks, or 8 weeks or more,e.g., 3 months, 4 months, 5 months, or 6 months or more.

In some instances, the methods result in a change in expression levelsof one or more genes in one or more tissues of the host, e.g., ascompared to a suitable control (such as described in the Experimentalsection, below). The change in expression level of a given gene may be0.5 fold or greater, such as 1.0 fold or greater, including 1.5 fold orgreater. The tissue may vary, and in some instances is nervous systemtissue, e.g., central nervous system tissue, including brain tissue,e.g., hippocampal tissue. In some instances, the modulation ofhippocampal gene expression is manifested as enhanced hippocampalplasticity, e.g., as compared to a suitable control.

In some instances, treatment results in an enhancement in the levels ofone or more proteins in one or more tissues of the host, e.g., ascompared to a suitable control (such as described in the Experimentalsection, below). The change in protein level of a given protein may be0.5 fold or greater, such as 1.0 fold or greater, including 1.5 fold orgreater, where in some instances the level may approach that of ahealthy wild-type level, e.g., within 50% or less, such as 25% or less,including 10% or less, e.g., 5% or less of the healthy wild-type level.The tissue may vary, and in some instances is nervous system tissue,e.g., central nervous system tissue, including brain tissue, e.g.,hippocampal tissue.

In some instances, the methods result in one or more structural changesin one or more tissues. The tissue may vary, and in some instances isnervous system tissue, e.g., central nervous system tissue, includingbrain tissue, e.g., hippocampal tissue. Structure changes of interestinclude an increase in dendritic spine density of mature neurons in thedentate gyrus (DG) of the hippocampus, e.g., as compared to a suitablecontrol. In some instances, the modulation of hippocampal structure ismanifested as enhanced synapse formation, e.g., as compared to asuitable control. In some instances, the methods may result in anenhancement of long term potentiation, e.g., as compared to a suitablecontrol.

In some instances, practice of the methods, e.g., as described above,results in an increase in neurogenesis in the adult mammal. The increasemay be identified in a number of different ways, e.g., as describedbelow in the Experimental section. In some instances, the increase inneurogenesis manifests as an increase the amount of Dcx-positiveimmature neurons, e.g., where the increase may be 2-fold or greater. Insome instances, the increase in neurogenesis manifests as an increase inthe number of BrdU/NeuN positive cells, where the increase may be 2-foldor greater.

In some instances, the methods result in enhancement in learning andmemory, e.g., as compared to a suitable control. Enhancement in learningand memory may be evaluated in a number of different ways, e.g., thecontextual fear conditioning and/or radial arm water maze (RAWM)paradigms described in the experimental section, below. When measured bycontextual fear conditioning, treatment results in some instances inincreased freezing in contextual, but not cued, memory testing. Whenmeasured by RAWM, treatment results in some instances in enhancedlearning and memory for platform location during the testing phase ofthe task. In some instances, treatment is manifested as enhancedcognitive improvement in hippocampal-dependent learning and memory,e.g., as compared to a suitable control.

In some embodiments, eotaxin-1/CCR3 interaction modulation, e.g., asdescribed above, may be performed in conjunction with an active agenthaving activity suitable to treat aging-associated cognitive impairment.For example, a number of active agents have been shown to have someefficacy in treating the cognitive symptoms of Alzheimer's disease(e.g., memory loss, confusion, and problems with thinking andreasoning), e.g., cholinesterase inhibitors (e.g., Donepezil,Rivastigmine, Galantamine, Tacrine), Memantine, and Vitamin E. Asanother example, a number of agents have been shown to have someefficacy in treating behavioral or psychiatric symptoms of Alzheimer'sDisease, e.g., citalopram (Celexa), fluoxetine (Prozac), paroxeine(Paxil), sertraline (Zoloft), trazodone (Desyrel), lorazepam (Ativan),oxazepam (Serax), aripiprazole (Abilify), clozapine (Clozaril),haloperidol (Haldol), olanzapine (Zyprexa), quetiapine (Seroquel),risperidone (Risperdal), and ziprasidone (Geodon).

In some aspects of the subject methods, the method further comprises thestep of measuring cognition and/or synaptic plasticity after treatment,e.g., using the methods described herein or known in the art, anddetermining that the rate of cognitive decline or loss of synapticplasticity have been reduced and/or that cognitive ability or synapticplasticity have improved in the individual. In some such instances, thedetermination is made by comparing the results of the cognition orsynaptic plasticity test to the results of the test performed on thesame individual at an earlier time, e.g., 2 weeks earlier, 1 monthearlier, 2 months earlier, 3 months earlier, 6 months earlier, 1 yearearlier, 2 years earlier, 5 years earlier, or 10 years earlier, or more.

In some embodiments, the subject methods further include diagnosing anindividual as having a cognitive impairment, e.g., using the methodsdescribed herein or known in the art for measuring cognition andsynaptic plasticity, prior to administering the subjectplasma-comprising blood product. In some instances, the diagnosing willcomprise measuring cognition and/or synaptic plasticity and comparingthe results of the cognition or synaptic plasticity test to one or morereferences, e.g., a positive control and/or a negative control. Forexample, the reference may be the results of the test performed by oneor more age-matched individuals that experience aging-associatedcognitive impairments (i.e., positive controls) or that do notexperience aging-associated cognitive impairments (i.e., negativecontrols). As another example, the reference may be the results of thetest performed by the same individual at an earlier time, e.g., 2 weeksearlier, 1 month earlier, 2 months earlier, 3 months earlier, 6 monthsearlier, 1 year earlier, 2 years earlier, 5 years earlier, or 10 yearsearlier, or more.

In some embodiments, the subject methods further include diagnosing anindividual as having an aging-associated disorder, e.g., Alzheimer'sdisease, Parkinson's disease, frontotemporal dementia, progressivesupranuclear palsy, Huntington's disease, amyotrophic lateral sclerosis,spinal muscular atrophy, multiple sclerosis, multi-system atrophy,glaucoma, ataxias, myotonic dystrophy, dementia, and the like. Methodsfor diagnosing such aging-associated disorders are well-known in theart, any of which may be used by the ordinarily skilled artisan indiagnosing the individual. In some embodiments, the subject methodsfurther comprise both diagnosing an individual as having anaging-associated disorder and as having a cognitive impairment.

Utility

The subject methods find use in treating, including preventing,aging-associated impairments and conditions associated therewith, suchas impairments in the cognitive ability of individuals. Individualssuffering from or at risk of developing an aging-associated cognitiveimpairments include individuals that are about 50 years old or older,e.g., 60 years old or older, 70 years old or older, 80 years old orolder, 90 years old or older, and usually no older than 100 years old,i.e., between the ages of about 50 and 100, e.g., 50, 55, 60, 65, 70,75, 80, 85, 90, 95 or about 100 years old, and are suffering fromcognitive impairment associated with natural aging process, e.g., mildcognitive impairment (M.C.I.); and individuals that are about 50 yearsold or older, e.g., 60 years old or older, 70 years old or older, 80years old or older, 90 years old or older, and usually no older than 100years old, i.e., between the ages of about 50 and 90, e.g., 50, 55, 60,65, 70, 75, 80, 85, 90, 95 or about 100 years old, that have not yetbegun to show symptoms of cognitive impairment. Examples of cognitiveimpairments that are due to natural aging include the following:

Mild cognitive impairment (M.C.I.) is a modest disruption of cognitionthat manifests as problems with memory or other mental functions such asplanning, following instructions, or making decisions that have worsenedover time while overall mental function and daily activities are notimpaired. Thus, although significant neuronal death does not typicallyoccur, neurons in the aging brain are vulnerable to sub-lethalage-related alterations in structure, synaptic integrity, and molecularprocessing at the synapse, all of which impair cognitive function.

Individuals suffering from or at risk of developing an aging-associatedcognitive impairment that will benefit from treatment with the subjectplasma-comprising blood product, e.g., by the methods disclosed herein,also include individuals of any age that are suffering from a cognitiveimpairment due to an aging-associated disorder; and individuals of anyage that have been diagnosed with an aging-associated disorder that istypically accompanied by cognitive impairment, where the individual hasnot yet begun to present with symptoms of cognitive impairment. Examplesof such aging-associated disorders include the following:

Alzheimer's disease (AD). Alzheimer's disease is a progressive,inexorable loss of cognitive function associated with an excessivenumber of senile plaques in the cerebral cortex and subcortical graymatter, which also contains b-amyloid and neurofibrillary tanglesconsisting of tau protein. The common form affects persons >60 yr old,and its incidence increases as age advances. It accounts for more than65% of the dementias in the elderly.

The cause of Alzheimer's disease is not known. The disease runs infamilies in about 15 to 20% of cases. The remaining, so-called sporadiccases have some genetic determinants. The disease has an autosomaldominant genetic pattern in most early-onset and some late-onset casesbut a variable late-life penetrance. Environmental factors are the focusof active investigation.

In the course of the disease, synapses, and ultimately neurons are lostwithin the cerebral cortex, hippocampus, and subcortical structures(including selective cell loss in the nucleus basalis of Meynert), locuscaeruleus, and nucleus raphae dorsalis. Cerebral glucose use andperfusion is reduced in some areas of the brain (parietal lobe andtemporal cortices in early-stage disease, prefrontal cortex inlate-stage disease). Neuritic or senile plaques (composed of neurites,astrocytes, and glial cells around an amyloid core) and neurofibrillarytangles (composed of paired helical filaments) play a role in thepathogenesis of Alzheimer's disease. Senile plaques and neurofibrillarytangles occur with normal aging, but they are much more prevalent inpersons with Alzheimer's disease.

Parkinson's Disease. Parkinson's Disease (PD) is an idiopathic, slowlyprogressive, degenerative CNS disorder characterized by slow anddecreased movement, muscular rigidity, resting tremor, and posturalinstability. Originally considered primarily a motor disorder, PD is nowrecognized to also affect cognition, behavior, sleep, autonomicfunction, and sensory function. The most common cognitive impairmentsinclude an impairment in attention and concentration, working memory,executive function, producing language, and visuospatial function.

In primary Parkinson's disease, the pigmented neurons of the substantianigra, locus caeruleus, and other brain stem dopaminergic cell groupsare lost. The cause is not known. The loss of substantia nigra neurons,which project to the caudate nucleus and putamen, results in depletionof the neurotransmitter dopamine in these areas. Onset is generallyafter age 40, with increasing incidence in older age groups.

Secondary parkinsonism results from loss of or interference with theaction of dopamine in the basal ganglia due to other idiopathicdegenerative diseases, drugs, or exogenous toxins. The most common causeof secondary parkinsonism is ingestion of antipsychotic drugs orreserpine, which produce parkinsonism by blocking dopamine receptors.Less common causes include carbon monoxide or manganese poisoning,hydrocephalus, structural lesions (tumors, infarcts affecting themidbrain or basal ganglia), subdural hematoma, and degenerativedisorders, including striatonigral degeneration.

Frontotemporal dementia. Frontotemporal dementia (FTD) is a conditionresulting from the progressive deterioration of the frontal lobe of thebrain. Over time, the degeneration may advance to the temporal lobe.Second only to Alzheimer's disease (AD) in prevalence, FTD accounts for20% of pre-senile dementia cases. Symptoms are classified into threegroups based on the functions of the frontal and temporal lobesaffected: Behavioural variant FTD (bvFTD), with symptoms includelethargy and aspontaneity on the one hand, and disinhibition on theother; progressive nonfluent aphasia (PNFA), in which a breakdown inspeech fluency due to articulation difficulty, phonological and/orsyntactic errors is observed but word comprehension is preserved; andsemantic dementia (SD), in which patients remain fluent with normalphonology and syntax but have increasing difficulty with naming and wordcomprehension. Other cognitive symptoms common to all FTD patientsinclude an impairment in executive function and ability to focus. Othercognitive abilities, including perception, spatial skills, memory andpraxis typically remain intact. FTD can be diagnosed by observation ofreveal frontal lobe and/or anterior temporal lobe atrophy in structuralMRI scans.

A number of forms of FTD exist, any of which may be treated or preventedusing the subject methods and compositions. For example, one form offrontotemporal dementia is Semantic Dementia (SD). SD is characterizedby a loss of semantic memory in both the verbal and non-verbal domains.SD patients often present with the complaint of word-findingdifficulties. Clinical signs include fluent aphasia, anomia, impairedcomprehension of word meaning, and associative visual agnosia (theinability to match semantically related pictures or objects). As thedisease progresses, behavioral and personality changes are often seensimilar to those seen in frontotemporal dementia although cases havebeen described of ‘pure’ semantic dementia with few late behavioralsymptoms. Structural MRI imaging shows a characteristic pattern ofatrophy in the temporal lobes (predominantly on the left), with inferiorgreater than superior involvement and anterior temporal lobe atrophygreater than posterior.

As another example, another form of frontotemporal dementia is Pick'sdisease (PiD, also PcD). A defining characteristic of the disease isbuild-up of tau proteins in neurons, accumulating into silver-staining,spherical aggregations known as “Pick bodies”. Symptoms include loss ofspeech (aphasia) and dementia. Patients with orbitofrontal dysfunctioncan become aggressive and socially inappropriate. They may steal ordemonstrate obsessive or repetitive stereotyped behaviors. Patients withdorsomedial or dorsolateral frontal dysfunction may demonstrate a lackof concern, apathy, or decreased spontaneity. Patients can demonstratean absence of self-monitoring, abnormal self-awareness, and an inabilityto appreciate meaning. Patients with gray matter loss in the bilateralposterolateral orbitofrontal cortex and right anterior insula maydemonstrate changes in eating behaviors, such as a pathologic sweettooth. Patients with more focal gray matter loss in the anterolateralorbitofrontal cortex may develop hyperphagia. While some of the symptomscan initially be alleviated, the disease progresses and patients oftendie within two to ten years.

Huntington's disease. Huntington's disease (HD) is a hereditaryprogressive neurodegenerative disorder characterized by the developmentof emotional, behavioral, and psychiatric abnormalities; loss ofintellectual or cognitive functioning; and movement abnormalities (motordisturbances). The classic signs of HD include the development ofchorea—involuntary, rapid, irregular, jerky movements that may affectthe face, arms, legs, or trunk—as well as cognitive decline includingthe gradual loss of thought processing and acquired intellectualabilities. There may be impairment of memory, abstract thinking, andjudgment; improper perceptions of time, place, or identity(disorientation); increased agitation; and personality changes(personality disintegration). Although symptoms typically become evidentduring the fourth or fifth decades of life, the age at onset is variableand ranges from early childhood to late adulthood (e.g., 70s or 80s).

HD is transmitted within families as an autosomal dominant trait. Thedisorder occurs as the result of abnormally long sequences or “repeats”of coded instructions within a gene on chromosome 4 (4p16.3). Theprogressive loss of nervous system function associated with HD resultsfrom loss of neurons in certain areas of the brain, including the basalganglia and cerebral cortex.

Amyotrophic lateral sclerosis. Amyotrophic lateral sclerosis (ALS) is arapidly progressive, invariably fatal neurological disease that attacksmotor neurons. Muscular weakness and atrophy and signs of anterior horncell dysfunction are initially noted most often in the hands and lessoften in the feet. The site of onset is random, and progression isasymmetric. Cramps are common and may precede weakness. Rarely, apatient survives 30 years; 50% die within 3 years of onset, 20% live 5years, and 10% live 10 years. Diagnostic features include onset duringmiddle or late adult life and progressive, generalized motor involvementwithout sensory abnormalities. Nerve conduction velocities are normaluntil late in the disease. Recent studies have documented thepresentation of cognitive impairments as well, particularly a reductionin immediate verbal memory, visual memory, language, and executivefunction.

A decrease in cell body area, number of synapses and total synapticlength has been reported in even normal-appearing neurons of the ALSpatients. It has been suggested that when the plasticity of the activezone reaches its limit, a continuing loss of synapses can lead tofunctional impairment. Promoting the formation or new synapses orpreventing synapse loss may maintain neuron function in these patients.

Multiple Sclerosis. Multiple Sclerosis (MS) is characterized by varioussymptoms and signs of CNS dysfunction, with remissions and recurringexacerbations. The most common presenting symptoms are paresthesias inone or more extremities, in the trunk, or on one side of the face;weakness or clumsiness of a leg or hand; or visual disturbances, e.g.,partial blindness and pain in one eye (retrobulbar optic neuritis),dimness of vision, or scotomas. Common cognitive impairments includeimpairments in memory (acquiring, retaining, and retrieving newinformation), attention and concentration (particularly dividedattention), information processing, executive functions, visuospatialfunctions, and verbal fluency. Common early symptoms are ocular palsyresulting in double vision (diplopia), transient weakness of one or moreextremities, slight stiffness or unusual fatigability of a limb, minorgait disturbances, difficulty with bladder control, vertigo, and mildemotional disturbances; all indicate scattered CNS involvement and oftenoccur months or years before the disease is recognized. Excess heat mayaccentuate symptoms and signs.

The course is highly varied, unpredictable, and, in most patients,remittent. At first, months or years of remission may separate episodes,especially when the disease begins with retrobulbar optic neuritis.However, some patients have frequent attacks and are rapidlyincapacitated; for a few the course can be rapidly progressive.

Glaucoma. Glaucoma is a common neurodegenerative disease that affectsretinal ganglion cells (RGCs). Evidence supports the existence ofcompartmentalized degeneration programs in synapses and dendrites,including in RGCs. Recent evidence also indicates a correlation betweencognitive impairment in older adults and glaucoma (Yochim B P, et al.Prevalence of cognitive impairment, depression, and anxiety symptomsamong older adults with glaucoma. J Glaucoma. 2012; 21(4):250-254).

Macular degeneration. Macular degeneration is a clinical term that isused to describe a family of diseases that are all characterized by aprogressive loss of central vision associated with abnormalities ofBruch's membrane, the choroid, the neural retina and/or the retinalpigment epithelium. These disorders include very common conditions thataffect older subjects—such as Age-related macular degeneration (AMD) aswell as rarer, earlier-onset dystrophies that in some cases can bedetected in the first decade of life. Other maculopathies include NorthCarolina macular dystrophy, Sorsby's fundus dystrophy, Stargardt'sdisease, pattern dystrophy, Best disease and Malattia leventinese.

AMD is the leading cause of permanent vision loss for individuals overage 65, currently affecting approximately 15 million Americans. AMDaffects light-sensitive photoreceptor cells and pigmented epithelialcells in the macula, the center of the retina of the eye. While it maynot cause total blindness, the disease destroys central vision, makingreading, watching electronic monitor screens and driving impossible. Ithas no documented cure, has never demonstrated spontaneous remission andeffective treatments are very limited.

The retina is a complicated network of nerve cells that changes lightinto nerve impulses that travel to the brain where they are interpretedas visual images. The central part of the retina, called the macula, isresponsible for vision that is needed for reading and other detailedwork. Damage to the macula results in poor vision. The most commondisease process that affects the macula is AMD. In patients with AMD,retinal photoreceptor and pigment epithelial cells in the macula dieover the course of several years. The cell death and gradual visual lossusually do not begin until age 60 or older, hence the name age-relatedmacular degeneration.

There are two types of AMD: dry macular degeneration and wet maculardegeneration. Dry macular degeneration, although more common, typicallyresults in a less severe, more gradual loss of vision. Patients who areaffected by dry AMD have gradual loss of central vision due to the deathof photoreceptor cells and their close associates, retinal pigmentedepithelial (RPE) cells, with deposition of a complex waxy amyloidmixture, termed ‘drusen’. Photoreceptors, the cells in the retina thatactually ‘see’ light, are essential for vision. Macrophagic RPE cellsare necessary for photoreceptor survival, function and renewal. Patientswith wet macular degeneration develop new blood vessels under theretina. As the photoreceptor and RPE cells slowly degenerate, there is atendency for blood vessels to grow from their normal location in thechoroid into an abnormal location beneath the retina. This abnormal newblood vessel growth is called choroidal neovascularization (CNV). Theabnormal blood vessels leak and bleed, causing hemorrhage, swelling,scar tissue, and severe loss of central vision. Only 10% of patientswith AMD have the wet type, but it is responsible for 90% of allblindness resulting from AMD.

Myotonic dystrophy. Myotonic dystrophy (DM) is an autosomal dominantmultisystem disorder characterized by dystrophic muscle weakness andmyotonia. The molecular defect is an expanded trinucleotide (CTG) repeatin the 3″ untranslated region of the myotonin-protein kinase gene onchromosome 19q. Symptoms can occur at any age, and the range of clinicalseverity is broad. Myotonia is prominent in the hand muscles, and ptosisis common even in mild cases. In severe cases, marked peripheralmuscular weakness occurs, often with cataracts, premature balding,hatchet facies, cardiac arrhythmias, testicular atrophy, and endocrineabnormalities (e.g., diabetes mellitus). Mental retardation is common insevere congenital forms, while an aging-related decline of frontal andtemporal cognitive functions, particularly language and executivefunctions, is observed in milder adult forms of the disorder. Severelyaffected persons die by their early 50s.

Dementia. Dementia describes class of disorders having symptomsaffecting thinking and social abilities severely enough to interferewith daily functioning. Other instances of dementia in addition to thedementia observed in later stages of the aging-associated disordersdiscussed above include vascular dementia, and dementia with Lewybodies, described below.

In vascular dementia, or “multi-infarct dementia”, cognitive impairmentis caused by problems in supply of blood to the brain, typically by aseries of minor strokes, or sometimes, one large stroke preceded orfollowed by other smaller strokes. Vascular lesions can be the result ofdiffuse cerebrovascular disease, such as small vessel disease, or focallesions, or both. Patients suffering from vascular dementia present withcognitive impairment, acutely or subacutely, after an acutecerebrovascular event, after which progressive cognitive decline isobserved.

Cognitive impairments are similar to those observed in Alzheimer'sdisease, including impairments in language, memory, complex visualprocessing, or executive function, although the related changes in thebrain are not due to AD pathology but to chronic reduced blood flow inthe brain, eventually resulting in dementia. Single photon emissioncomputed tomography (SPECT) and positron emission tomography (PET)neuroimaging may be used to confirm a diagnosis of multi-infarctdementia in conjunction with evaluations involving mental statusexamination.

Dementia with Lewy bodies (DLB, also known under a variety of othernames including Lewy body dementia, diffuse Lewy body disease, corticalLewy body disease, and senile dementia of Lewy type) is a type ofdementia characterized anatomically by the presence of Lewy bodies(clumps of alpha-synuclein and ubiquitin protein) in neurons, detectablein post mortem brain histology. Its primary feature is cognitivedecline, particularly of executive functioning. Alertness and short termmemory will rise and fall. Persistent or recurring visual hallucinationswith vivid and detailed pictures are often an early diagnostic symptom.DLB it is often confused in its early stages with Alzheimer's diseaseand/or vascular dementia, although, where Alzheimer's disease usuallybegins quite gradually, DLB often has a rapid or acute onset. DLBsymptoms also include motor symptoms similar to those of Parkinson's.DLB is distinguished from the dementia that sometimes occurs inParkinson's disease by the time frame in which dementia symptoms appearrelative to Parkinson symptoms. Parkinson's disease with dementia (PDD)would be the diagnosis when dementia onset is more than a year after theonset of Parkinson's. DLB is diagnosed when cognitive symptoms begin atthe same time or within a year of Parkinson symptoms.

Progressive supranuclear palsy. Progressive supranuclear palsy (PSP) isa brain disorder that causes serious and progressive problems withcontrol of gait and balance, along with complex eye movement andthinking problems. One of the classic signs of the disease is aninability to aim the eyes properly, which occurs because of lesions inthe area of the brain that coordinates eye movements. Some individualsdescribe this effect as a blurring. Affected individuals often showalterations of mood and behavior, including depression and apathy aswell as progressive mild dementia. The disorder's long name indicatesthat the disease begins slowly and continues to get worse (progressive),and causes weakness (palsy) by damaging certain parts of the brain abovepea-sized structures called nuclei that control eye movements(supranuclear). PSP was first described as a distinct disorder in 1964,when three scientists published a paper that distinguished the conditionfrom Parkinson's disease. It is sometimes referred to asSteele-Richardson-Olszewski syndrome, reflecting the combined names ofthe scientists who defined the disorder. Although PSP gets progressivelyworse, no one dies from PSP itself.

Ataxia. People with ataxia have problems with coordination because partsof the nervous system that control movement and balance are affected.Ataxia may affect the fingers, hands, arms, legs, body, speech, and eyemovements. The word ataxia is often used to describe a symptom ofincoordination which can be associated with infections, injuries, otherdiseases, or degenerative changes in the central nervous system. Ataxiais also used to denote a group of specific degenerative diseases of thenervous system called the hereditary and sporadic ataxias which are theNational Ataxia Foundation's primary emphases.

Multiple-system atrophy. Multiple-system atrophy (MSA) is a degenerativeneurological disorder. MSA is associated with the degeneration of nervecells in specific areas of the brain. This cell degeneration causesproblems with movement, balance, and other autonomic functions of thebody such as bladder control or blood-pressure regulation. The cause ofMSA is unknown and no specific risk factors have been identified. Around55% of cases occur in men, with typical age of onset in the late 50s toearly 60s. MSA often presents with some of the same symptoms asParkinson's disease. However, MSA patients generally show minimal if anyresponse to the dopamine medications used for Parkinson's.

In some embodiments, the subject methods and compositions find use inslowing the progression of aging-associated cognitive impairment. Inother words, cognitive abilities in the individual will decline moreslowly following treatment by the disclosed methods than prior to or inthe absence of treatment by the disclosed methods. In some suchinstances, the subject methods of treatment include measuring theprogression of cognitive decline after treatment, and determining thatthe progression of cognitive decline is reduced. In some such instances,the determination is made by comparing to a reference, e.g., the rate ofcognitive decline in the individual prior to treatment, e.g., asdetermined by measuring cognition prior at two or more time points priorto administration of the subject blood product.

The subject methods and compositions also find use in stabilizing thecognitive abilities of an individual, e.g., an individual suffering fromaging-associated cognitive decline or an individual at risk of sufferingfrom aging-associated cognitive decline. For example, the individual maydemonstrate some aging-associated cognitive impairment, and progressionof cognitive impairment observed prior to treatment with the disclosedmethods will be halted following treatment by the disclosed methods. Asanother example, the individual may be at risk for developing anaging-associated cognitive decline (e.g., the individual may be aged 50years old or older, or may have been diagnosed with an aging-associateddisorder), and the cognitive abilities of the individual aresubstantially unchanged, i.e., no cognitive decline can be detected,following treatment by the disclosed methods as compared to prior totreatment with the disclosed methods.

The subject methods and compositions also find use in reducing cognitiveimpairment in an individual suffering from an aging-associated cognitiveimpairment. In other words, cognitive ability is improved in theindividual following treatment by the subject methods. For example, thecognitive ability in the individual is increased, e.g., by 2-fold ormore, 5-fold or more, 10-fold or more, 15-fold or more, 20-fold or more,30-fold or more, or 40-fold or more, including 50-fold or more, 60-foldor more, 70-fold or more, 80-fold or more, 90-fold or more, or 100-foldor more, following treatment by the subject methods relative to thecognitive ability that is observed in the individual prior to treatmentby the subject methods. In some instances, treatment by the subjectmethods and compositions restores the cognitive ability in theindividual suffering from aging-associated cognitive decline, e.g., totheir level when the individual was about 40 years old or less. In otherwords, cognitive impairment is abrogated.

The subject methods and compositions also find use in reducing cognitiveimpairment in an individual suffering from cognitive decline as aconsequence of systemic inflammation, radiation, chemotherapy, frailty,and kidney dysfunction. The subject methods and compositions also finduse in reducing, if not preventing, age-associated brain inflammation,neurodegeneration and cognitive decline.

Reagents, Devices and Kits

Also provided are reagents, devices and kits thereof for practicing oneor more of the above-described methods. The subject reagents, devicesand kits thereof may vary greatly. Reagents and devices of interestinclude those mentioned above with respect to the methods of modulatingeotaxin-1/CCR3 interaction in an adult mammal.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, portable flash drive, etc., on which the informationhas been recorded. Yet another means that may be present is a websiteaddress which may be used via the internet to access the information ata removed site. Any convenient means may be present in the kits.

The following examples are provided by way of illustration and not byway of limitation.

Experimental I. Materials and Methods A. Summary of Methods

C57BL/6 (Jackson Laboratory), C57BL/6 aged mice (National Institutes ofAging), Dcx-Luc26, and C57BL/6J-Act-GFP (Jackson Laboratory). For all invivo pharmacological and behavioral studies young (2-3 months) wild typeC57BL/6 male mice were used. All animal use was in accordance withinstitutional guidelines approved by the VA Palo Alto Committee onAnimal Research. Parabiosis surgery followed previously describedprocedures (Monje et al., Science (2003) 302: 1760-1765) with theaddition that peritonea between animals were surgically connected.Immunohistochemistry was performed on free-floating sections followingstandard published techniques (Luo et al., J Clin Invest (2007) 117:3306-3315). Hippocampal slice extracellular electrophysiology wasperformed as previously described (Xie & Smart, Pflugers Arch (1994)427: 481-486). Spatial learning and memory was assayed with the radialarm water maze (RAWM) paradigm as previously published (Alamed et al.,Nat Protoc (2006)1:1671-1679). Mouse plasma was prepared bycentrifugation and systemically administered via intravenous injections.Relative plasma concentrations of cytokines and signaling molecules inmice and humans were measured using antibody-based multipleximmunoassays at Rules Based Medicine, Inc. Human plasma and CSF sampleswere obtained from academic centers and informed consent was obtainedfrom human subjects according to the institutional review boardguidelines at the respective centers. Recombinant murine CCL11 (R&DSystems), rat IgG2a neutralizing antibody against mouse CCL11 (R&DSystems), and control rat IgG2a (R&D Systems) were administered eithersystemically by intraperitoneal injection or locally by unilateralstereotaxic injection into the dentate gyrus of the hippocampus.Statistical analysis was performed with Prism 5.0 software (GraphPadSoftware). Plasma protein correlations in the aging samples wereanalyzed with the Significance Analysis of Microarray software (SAM 3.00algorithm).

B. Mice

The following mouse lines were used: C57BL/6 (The Jackson Laboratory),C57BL/6 aged mice (National Institutes of Aging), Dcx-Luc mice(Couillard-Despres et al., Mol Imaging (2008) 7:28-34), andC57BL/6J-Act-GFP (Jackson Laboratory). For all in vivo pharmacologicaland behavioral studies young (2-3 months) wild type C57BL/6 male micewere used. Mice were housed under specific pathogen-free conditionsunder a 12 h light-dark cycle and all animal handling and use was inaccordance with institutional guidelines approved by the VA Palo AltoCommittee on Animal Research. All experiments were done in a randomizedand blinded fashion.

C. Immunohistochemistry

Tissue processing and immunohistochemistry was performed onfree-floating sections following standard published techniques (Luo etal., J Clin Invest (2007) 117:3306-3315). Briefly, mice wereanesthetized with 400 mg/kg chloral hydrate (Sigma-Aldrich) andtranscardially perfused with 0.9% saline Brains were removed and fixedin phosphate-buffered 4% paraformaldehyde, pH 7.4, at 4° C. for 48 hbefore they were sunk through 30% sucrose for cryoprotection. Brainswere then sectioned coronally at 40 μm with a cryomicrotome (LeicaCamera, Inc.) and stored in cryoprotective medium. Primary antibodieswere: goat anti-Dcx (1:500; Santa Cruz Biotechnology), rat anti-BrdU(1:5000, Accurate Chemical and Scientific Corp.), goat anti-Sox2 (1:200;Santa Cruz), mouse anti-NeuN (1:1000, Chemicon), mouse anti-GFAP(1:1500, DAKO), and mouse anti-CD68 (1:50, Serotec). After overnightincubation, primary antibody staining was revealed using biotinylatedsecondary antibodies and the ABC kit (Vector) with Diaminobenzidine(DAB, Sigma-Aldrich) or fluorescence conjugated secondary antibodies.For BrdU labeling, brain sections were pre-treated with 2N HCl at 37° C.for 30 min before incubation with primary antibody. For double-labelimmunofluorescence of BrdU/NeuN or BrdU/GFAP, sections were incubatedovernight with rat anti-BrdU, rinsed, and incubated for 1 hr with donkeyanti-rat antibody (2.5 μg/ml, Vector) before they were stained withmouse anti-NeuN antibody.

To estimate the total number of Dcx or Sox2 positive cells per DGimmunopositive cells in the granule cell and subgranular cell layer ofthe DG were counted in every sixth coronal hemibrain section through thehippocampus and multiplied by 12.

D. BrdU Administration and Quantification of BrdU-Positive Cells

50 mg/kg of BrdU was injected intraperitoneally into mice once a day for6 days, and mice were sacrificed 28 days later or injected daily for 3days before sacrifice. To estimate the total number of BrdU-positivecells in the brain, we performed DAB staining for BrdU on every sixthhemibrain section. The number of BrdU+ cells in the granule cell andsubgranular cell layer of the DG were counted and multiplied by 12 toestimate the total number of BrdU-positive cells in the entire DG. Todetermine the fate of dividing cells a total of 200 BrdU-positive cellsacross 4-6 sections per mouse were analyzed by confocal microscopy forco-expression with NeuN and GFAP. The number of double-positive cellswas expressed as a percentage of BrdU-positive cells.

E. Parabiosis and Flow Cytometry

Parabiosis surgery followed previously described procedures (Conboy etal., Nature (2005) 433: 760-764). Pairs of mice were anesthetized andprepared for surgery. Mirror-image incisions at the left and rightflanks, respectively, were made through the skin. Shorter incisions weremade through the abdominal wall. The peritoneal openings of the adjacentparabionts were sutured together. Elbow and knee joints from eachparabiont were sutured together and the skin of each mouse was stapled(9 mm Autoclip, Clay Adams) to the skin of the adjacent parabiont. Eachmouse was injected subcutaneously with Baytril antibiotic and Buprenexas directed for pain and monitored during recovery. Flow cytometricanalysis was done on fixed and permeabilized blood plasma cells from GFPand non-GFP parabionts. Approximately 40-60% of cells in the blood ofeither parabiont were GFP-positive two weeks after parabiosis surgery.We observed 70-80% survival rate in parabionts five weeks postparabiosis surgery.

F. Extracellular Electrophysiology

Acute hippocampal slices (400 μm thick) were prepared from unpaired andyoung parabionts. Slices were maintained in artificial cerebrospinalfluid (ACSF) continuously oxygenated with 5% CO₂/95% O₂. ACSFcomposition was as follows: (in mM): NaCl 124.0; KCl 2.5; KH₂PO₄ 1.2;CaCl₂ 2.4; MgSO₄ 1.3; NaHCO₃ 26.0; glucose 10.0 (pH 7.4). Recordingswere performed with an Axopatch-2B amplifier and pClamp 10.2 software(Axon Instruments). Submerged slices were continuously perfused withoxygenated ACSF at a flow rate of 2 ml/min from a reservoir by gravityfeeding. Field potential (population spikes and EPSP) was recorded usingglass microelectrodes filled with ACSF (resistance: 4-8 M.OMEGA.).Biphasic current pulses (0.2 ms duration for one phase, 0.4 ms in total)were delivered in 10 s intervals through a concentric bipolarstimulating electrode (FHC, Inc.). No obvious synaptic depression orfacilitation was observed with this frequency stimulation. To recordfield population spikes in the dentate gyrus, the recording electrodewas placed in the lateral or medial side of the dorsal part of thedentate gyrus. The stimulating electrode was placed right above thehippocampal fissure to stimulate the perforant pathway fibers. Signalswere filtered at 1 KHz and digitized at 10 KHz. Tetanic stimulationconsisted of 2 trains of 100 pulses (0.4 ms pulse duration, 100 Hz)delivered with an inter-train interval of 5 seconds. The amplitude ofpopulation spike was measured from the initial phase of the negativewave. Up to five consecutive traces were averaged for each measurement.LTP was calculated as mean percentage change in the amplitude of thepopulation spike following high frequency stimulation relative to itsbasal amplitude.

G. Behavioral Assay

Spatial learning and memory was assessed using the radial arm water maze(RAWM) paradigm following the exact protocol described by Alamed et al.Nat Protoc (2006)1:1671-1679). Behavioral analysis was performed fornormal aging mice at young (2-3 months) and old (18 months) ages, foryoung adult mice (2-3 months) injected intravenously with plasmaisolated from young (3-4 months) and old (18-20 months) mice every threedays for 24 days, and for young adult mice (3-4 months) injectedintraperitoneally with murine recombinant CCL11 and PBS vehicle for fiveweeks. The goal arm location containing a platform remains constantthroughout the training and testing phase, while the start arm ischanged during each trial. On day one during the training phase, miceare trained for 15 trails, with trials alternating between a visible andhidden platform. On day two during the testing phase, mice are testedfor 15 trials with a hidden platform. Entry into an incorrect arm isscored as an error, and errors are averaged over training blocks (threeconsecutive trials). All studies were done by an investigator that wasblinded to the age or treatment of mice.

H. Plasma Collection and Proteomic Analysis

Mouse blood was collected into EDTA coated tubes via tail vein bleed,mandibular vein bleed, or intracardial bleed at time of sacrifice. EDTAplasma was generated by centrifugation of freshly collected blood andaliquots were stored at −80° C. until use. Human plasma and CSF sampleswere obtained from academic centers and subjects were chosen based onstandardized inclusion and exclusion criteria as previously described(Zhang et al., Am J Clin Pathol (2008)129: 526-529; Li et al., PLoS One4 (2009) (5), e5424) and outlined below. Mouse and human plasma sampleswere sent to Rules Based Medicine Inc., a fee-for-service provider,where the relative plasma concentrations of cytokines and signalingmolecules were measured using standard antibody-based multipleximmunoassays in a blinded fashion. All assays were developed andvalidated to Clinical Laboratory Standards Institute (formerly NCCLS)guidelines based upon the principles of immunoassay as described by themanufacturers.

I. CCL11, MSCF, Antibody, or Plasma Administration

Carrier free recombinant murine CCL11 dissolved in PBS (10 μg/kg; R&DSystems), carrier free recombinant MCSF dissolved in PBS (10 μg/kg;Biogen), rat IgG2a neutralizing antibody against mouse CCL11 (50 μg/ml;R&D Systems, Clone: 42285), and isotype matched control rat IgG2arecommended by the manufacturer (R&D Systems, Clone: 54447) wereadministered systemically via intraperitoneal injection over ten days onday 1, 4, 7, and 10. The same reagents (0.50 μl; 0.1 μg/μl) were alsoadministered stereotaxically into the DG of the hippocampus in someexperiments (coordinates from bregma: A=−2.0 mm and L=−1.8 mm, frombrain surface: H=−2.0 mm). Pooled mouse serum or plasma was collectedfrom 2-3-month-old (young) mice and 18-20-month-old (aged) mice byintracardial bleed at time of sacrifice. Serum was prepared from clottedblood collected without anticoagulants; plasma was prepared from bloodcollected with EDTA followed by centrifugation. Aliquots were stored at−80° C. until use. Prior to administration plasma was dialyzed in PBS toremove EDTA. Young adult mice were systemically treated with plasma (100μl) isolated from young or aged mice via intravenous injections everythree days for ten days.

J. In Vivo Bioluminescence Imaging

Bioluminescence was detected with the In Vivo Imaging System (IVISSpectrum; Caliper Life Science). Mice were injected intraperitoneallywith 150 mg/kg D-luciferin (Xenogen) 10 minutes before imaging andanesthetized with isofluorane during imaging. Photons emitted fromliving mice were acquired as photons/s/cm2/steridan (sr) usingLIVINGIMAGE software (version 3.5, Caliper) and integrated over 5minutes. For quantification a region of interest was manually selectedand kept constant for all experiments.

K. Cell Culture Assays

Mouse neural progenitor cells were isolated from C57BL/6 mice aspreviously described (Renault et al., Cell Stem Cell (2009) 5: 527-539).Brains from postnatal animals (1 day-old) were dissected to removeolfactory bulbs, cerebellum and brainstem. After removing superficialblood vessels forebrains were finely minced, digested for 30 minutes at37° C. in DMEM media containing 2.5 U/ml Papain (WorthingtonBiochemicals), 1 U/ml Dispase II (Boeringher Mannheim), and 250 U/mlDNase I (Worthington Biochemicals) and mechanically dissociated.NSC/progenitors were purified using a 65% Percoll gradient and plated onuncoated tissue culture dishes at a density of 105 cells/cm₂. NPCs werecultured under standard conditions in NeuroBasal A medium supplementedwith penicillin (100 U/ml), streptomycin (100 mg/ml), 2 mM L-glutamine,serum-free B27 supplement without vitamin A (Sigma-Aldrich), bFGF (20ng/ml) and EGF (20 ng/ml). Carrier free forms of murine recombinant CCL2(100 ng/ml; R&D Systemcs), murine recombinant CCL11 (100 ng/ml, R&DSystemcs), rat IgG2b neutralizing antibody against mouse CCL2 (10 ug/ml;R&D Systems, Clone: 123616), control rat IgG2b (10 μg/ml; R&D Systems,Clone: 141945), goat IgG neutralizing antibody against mouse CCL11 (10μg/ml; R&D Systems), and control goat IgG (10 μg/ml; R&D Systems) weredissolved in PBS and added to cell cultures under self-renewalconditions every other day following cell plating.

Human NTERA cells (Renault et al., Cell Stem Cell (2009) 5: 527-539)expressing eGFP under the doublecortin promoter were cultured understandard self-renewal and differentiation conditions (Couillard-Despreset al., BMC Neurosci (2008) 9: 31; Buckwalter et al., Am J Pathol (2006)169: 154-164). Carrier free forms of human recombinant CCL2 (100 ng/ml,R&D Systems), human recombinant CCL11 (100 ng/ml, R&D Systems), mouseIgG1 neutralizing antibody against human CCL11 (25 μg/ml; R&D Systems,Clone: 43911) and control mouse IgG1 (25 μg/ml; R&D Systems) were addedto cell cultures under differentiation conditions every other dayfollowing cell plating.

L. Data and Statistical Analysis

Data are expressed as mean±SEM. Statistical analysis was performed withPrism 5.0 software (Graph Pad Software). Means between two groups werecompared with two-tailed, unpaired Student's t test. Comparisons ofmeans from multiple groups with each other or against one control groupwere analyzed with 1-way ANOVA and Tukey-Kramer's or Dunnett's post hoctests, respectively. Plasma protein correlations in the aging sampleswere analyzed with the Significance Analysis of Microarray software (SAM3.00 algorithm, see, e.g., R. Hughey and A. Krogh, Technical ReportUCSC-CRL-95-7, University of California, Santa Cruz, Calif., January1995. (Last update prior to filing of application No. 61/298,998), TheSAM documentation.). Unsupervised cluster analysis was performed usingGene Cluster 3.0 software and node maps were produced using JavaTreeView 1.0.13 software.

II. Results and Discussion

During aging both regenerative capacity and cognitive functiondramatically deteriorate in the adult brain (Rando, T. A., Nature (2006)441:1080-1086; Rapp & Heindel, Curr Opin Neurol (1994) 7: 294-298).Interestingly, associated stem cell and cognitive impairments can beameliorated through systemic perturbations such as exercise (van Praaget al., J Neurosci (2005) 25: 8680-8685). Here, using heterochronicparabiosis we show that blood-borne factors present in the systemicmilieu can inhibit or rejuvenate adult neurogenesis in an age dependentfashion in mice. Accordingly, exposing a young animal to an old systemicenvironment, or to plasma from old mice, decreased synaptic plasticityand impaired spatial learning and memory. We identifychemokines—including CCL2/MCP-1 and CCL11/Eotaxin—whose plasma levelscorrelate with reduced neurogenesis in aged mice, and whose levels areincreased in plasma and cerebral spinal fluid of healthy aging humans.Finally, increasing peripheral chemokine levels in vivo in young micedecreased adult neurogenesis and impaired spatial learning and memory.Together our data indicate that the decline in neurogenesis, andcognitive impairments, observed during aging can be in part attributedto changes in blood-borne factors.

Stem cell activity decreases dramatically with age in tissues includingthe brain (Rando, T. A., Nature (2006) 441: 1080-1086). In the centralnervous system (CNS), aging results in a decline in adult neuralstem/progenitor cells (NPCs) and neurogenesis, with concomitantimpairments in cognitive functions (van Praag et al., J Neurosci (2005)25:8680-8685; Clelland et al., Science (2009) 325: 210-213). Adultneurogenesis occurs in local microenvironments, or neurogenic niches, inthe subventricular zone (SVZ) of the lateral ventricles and thesubgranular zone (SGZ) of the hippocampus (Gage, F. H., Science (2000)287:1433-1438; Alvarez-Buylla & Lim, Neuron (2004) 41: 683-686).Permissive cues within the neurogenic niche are thought to drive theproduction of new neurons and their subsequent integration into theneurocircuitry of the brain (Zhao et al., Cell (2008) 132: 645-660; vanPraag et al., Nature (2002) 415: 1030-1034), which directly contributesto cognitive processes including learning and memory (Clelland et al.,Science (2009) 325: 210-213; Deng et al., Nat Rev Neurosci (2010)11:339-350; Zhang et al., Nature (2008) 451: 1004-1007). Importantly,the neurogenic niche is localized around blood vessels (Shen et al.,Science (2004) 304: 1338-1340; Carpentier & Palmer, Neuron (2009) 64:79-92) that lack a classical blood-brain-barrier (BBB) (Shen et al.,Cell Stem Cell (2008) 3: 289-300; Tavazoie et al., Cell Stem Cell (2008)3:279-288; Currle & Gilbertson Cell Stem Cell (2008) 3: 234-236,allowing for potential communication with the systemic environment.Therefore, the possibility arises that diminished adult neurogenesisduring aging may be modulated by the balance of two independentforces—intrinsic CNS-derived cues previously reported (Renault et al.,Cell Stem Cell (2009) 5: 527-539; Molofsky et al., Nature (2006) 443:448-452; Lie et al., Nature (2005) 437: 1370-1375), and cues extrinsicto the CNS delivered by blood. We hypothesized that age-related systemicmolecular changes could cause a decline in neurogenesis and impaircognitive function during aging.

We first characterized the aging neurogenic niche by assessing cellularchanges in newly differentiated neurons, neural progenitors, microglia,and astrocytes in the dentate gyrus (DG) of the hippocampus in mice at6, 12, 18 and 24 months of age (FIGS. 5A-5D), and observed changesconsistent with a dramatic decrease in adult neurogenesis (van Praag etal., J Neurosci (2005) 25: 8680-8685) and a concomitant increase inneuroinflammation with age (Lucin & Wyss-Coray, Neuron (2009) 64:110-122). Additionally, we used a long-term potentiation (LTP) paradigmto examine synaptic plasticity, and detected lower LTP levels from theDG of old (18 months) versus young (3 months) animals (FIG. 6A). Lastly,we assessed hippocampal dependent spatial learning and memory using theradial arm water maze (RAWM) paradigm (Alamed et al., Nat Protoc (2006)1:1671-1679). During the training phase all animals showed learningcapacity for the task (FIG. 6B). However, old mice demonstrated impairedlearning and memory for platform location compared to young mice duringthe testing phase of the task (FIG. 6B), consistent with a decrease incognitive function during normal aging (Rapp & Heindel, Curr Opin Neurol(1994) 7:294-298).

To determine whether peripheral systemic factors contributed to thedecline in neurogenesis with age we utilized a model of parabiosis.Specifically, neurogenesis in the DG of the hippocampus was investigatedin the setting of isochronic (young-young (3-4 months) and old-old(18-20 months)) and heterochronic (young-old) parabiotic pairings (FIG.1A). Remarkably, the number of Doublecortin (Dcx)-positive newly bornneurons in young heterochronic parabionts decreased 20% compared toyoung isochronic parabionts (FIG. 1B). Likewise, BrdU-positive cells(FIG. 7B) and Sox2-positive progenitors (FIG. 7C) showed a similardecrease. In contrast, we observed a 3-fold increase in the number ofDcx-positive neurons (FIG. 10) and BrdU-positive cells (FIG. 7C) in theold heterochronic parabionts compared to isochronic old parabionts. Thenumber of Dcx-positive neurons between unpaired age-matched animals andisochronic animals showed no significant difference, indicating that theparabiosis procedure in it of itself did not account for the observedchanges (FIGS. 7D and 7E).

We also compared the neurite length of newly differentiated neurons inisochronic and heterochronic parabionts (FIGS. 1D, 1E). Youngheterochronic parabionts showed a 20% decrease in length compared toisochronic parabionts (FIG. 1D), while old heterochronic parabiontsdemonstrated a 40% increase in length compared to age-matched isochroniccontrols (FIG. 1E). Neurite length between unpaired age-matched animalsand isochronic parabionts showed no significant difference (FIG. 7F). Asa control, flow cytometry analysis confirmed a shared vasculature in asubset of parabiotic pairs, in which one parabiont was transgenic forgreen fluorescent protein (GFP, FIG. 8A-8D). Together our findingsindicate that global age-dependent systemic changes can modulateneurogenesis and neurite morphology in both the young and agedneurogenic niche, potentially contributing to the decline inregenerative capacity observed in the normal aging brain.

As previously reported by others (Ajami et al., Nat Neurosci (2007) 10:1538-1543), we rarely detected peripherally derived GFP cells in the CNSof wild-type mice when joined to GFP transgenic mice, and these numbersdid not differ between isochronic and heterochronic pairings (FIG. 8E),suggesting the observed effects are most likely mediated by solublefactors in plasma. To confirm that circulating factors within aged bloodcan contribute to reduced neurogenesis with age, we intravenouslyinjected plasma isolated from young (3-4 months) and old (18-22 months)mice into a cohort of young adult animals. The number of Dcx-positivecells in the DG decreased in animals receiving old plasma compared toanimals receiving young plasma (FIG. 2A), indicating that solublefactors present in old blood inhibit adult neurogenesis. To furtherinvestigate the functional effect of the aging systemic milieu on theyoung adult brain, extracellular electrophysiological recordings weredone on hippocampal slices prepared from young isochronic andheterochronic parabionts (FIG. 2B). We detected a decrease in LTP levelsin the medial and lateral DG of heterochronic parabionts compared toisochronic parabionts (FIG. 2C), indicating that age-related systemicchanges can elicit deficits in synaptic plasticity. Lastly, given thatLTP is considered a correlate of learning and memory (Bliss &Collingridge, Nature (1993) 361: 31-39), we sought to further evaluatethe physiological effect of circulating factors present in aged blood bytesting hippocampal dependent learning and memory using the RAWMparadigm in young adult mice intravenously injected with young or oldplasma (FIGS. 2D-2E). All mice showed similar spatial learning capacityduring the training phase (FIG. 2E). However, during the testing phaseanimals administered with old plasma demonstrated impaired learning andmemory for platform location, committing more errors in identifying thetarget arm compared to animals receiving young plasma (FIG. 2E).Collectively, these data indicate that factors present in aging bloodinhibit adult neurogenesis, and moreover functionally contribute toimpairments in synaptic plasticity and cognitive function.

Consistent with our cellular findings in the CNS, previous studiesfocusing on muscle stem cells also show that exposure of the aged stemcell niche to a young systemic environment through heterochronicparabiosis results in increased regeneration after muscle injury (Conboyet al., Nature (2005) 433 760-764). However, in these earlier modelsindividual circulating factors associated with either aging and tissuedegeneration, or tissue rejuvenation, have remained elusive. To identifysuch systemic factors, we employed a proteomic approach in which therelative levels of 66 cytokines, chemokines and other secreted signalingproteins were measured in the plasma of normal aging mice usingstandardized antibody-based immunoassays on microbeads. Usingmultivariate analysis, we identified seventeen blood borne proteins thatcorrelated with the age-related decline in neurogenesis during normalaging (FIGS. 3A, 9A-9B).

To identify systemic factors associated with heterochronic parabiosis,we analyzed plasma samples from young and old animals before and afterpairings in an independent proteomic screen using the Luminex platform.Comparison of young isochronic and heterochronic cohorts identifiedfourteen factors with a greater than 2-fold increase in expression inthe heterochronic parabionts (FIGS. 3A, FIG. 9C), while comparisonbetween old isochronic and heterochronic cohorts revealed four factorswhose expression levels decreased to less than 70% of that observed inisochronic parabionts (FIG. 9C). Interestingly, only five factors—CCL2,CCL11, CCL12, β2-microglobulin and Haptoglobin—were elevated in both oldunpaired and young heterochronic cohorts compared to young unpaired orisochronic cohorts (FIG. 3A). We observed a comparable increase in therelative levels of CCL2 and CCL11 in the plasma of mice during normalaging (FIGS. 3B-3C) and within young mice during heterochronicparabiosis (FIGS. 3D-3E).

To corroborate systemic changes in mice with changes occurring inhumans, we measured CCL2 and CCL11 in archived plasma and cerebrospinalfluid (CSF) samples from healthy individuals between 20 and 90 years ofage. Indeed, we detected an age-related increase in CCL2 and CCL11measured in both plasma (3F-3G) and CSF (FIGS. 3H-3I), suggesting thatthese age-related systemic molecular changes are conserved acrossspecies.

Having identified systemic factors associated with aging and decreasedneurogenesis, we tested their potential biological relevance in vivo. AsCCL2 had previously been linked to aging (Fumagalli & d'Adda di Fagagna,Nat Cell Biot (2009) 11: 921-923) and shown to regulate NPC functionafter brain injury (Belmadani et al., J Neurosci (2006) 26: 3182-3191),we decided to focus our study on CCL11, a chemokine involved in allergicresponses and not previously linked to aging, neurogenesis, orcognition. We administered recombinant murine CCL11 protein throughintraperitoneal injections into young adult mice and measured globalchanges in neurogenesis within the same mouse with a non-invasivebioluminescent imaging assay using Doublecortin-luciferase reporter mice(Couillard-Despres et al., Mol Imaging (2008) 7: 28-34). This systemicadministration of recombinant CCL11 caused a significant decrease in Dcxpromoter-dependent luciferase activity compared with mice receivingvehicle control indicating a decrease in the number of Dcx-expressingneuroblasts (FIGS. 4A-4B).

To confirm and expand upon this in vivo bioluminescent model, we nextinvestigated the effect of systemic CCL11 on adult hippocampalneurogenesis using immunohistochemical analysis. In an independentcohort of young wild type adult mice, we administered recombinant CCL11or vehicle alone, and in combination with either an anti-CCL11neutralizing antibody or an isotype control antibody throughintraperitoneal injections. The systemic administration of recombinantCCL11 induced an increase in CCL11 plasma levels (FIG. 10A), and causeda significant decrease in the number of Dcx-positive cells in the DGcompared to mice injected with vehicle control, consistent with in vivobioluminescent results (FIG. 4C). Importantly, this decrease inneurogenesis could be rescued by systemic neutralization of CCL11 (FIG.4C). Likewise, BrdU-positive cells also showed similar changes in cellnumber (FIG. 10C), and furthermore the percentage of cells expressingboth BrdU and NeuN decreased after systemic administration of CCL11(FIG. 4D). The percentage of cells expressing BrdU and GFAP did notsignificantly change (FIG. 10C). As a negative control we assayedneurogenesis in a cohort of young adult mice after systemicadministration of monocyte colony stimulating factor (MCSF), a proteinmeasured in both of our independent proteomic screens that did not showan age-dependent change in plasma levels or a correlation with reducedneurogenesis, and detected no change in Dcx-positive cells in the DG(FIGS. 11A-11C). Together, these data indicate that increasing thesystemic level of CCL11, an individual age-related factor identified inour unbiased screen, is sufficient to partially recapitulate some of theinhibitory effects on neurogenesis observed with aging and heterochronicparabiosis.

To investigate the possibility that age-related blood borne factors candirectly influence stem cell function, we used primary mouse NPCcultures as a model of neural stem cell activity. We observed a 50%decrease in the number of neurospheres formed after a four-day exposureof NPCs to aged mouse serum when compared to NPCs exposed to young serum(FIG. 12A). We then tested whether the identified chemokines could alsoexert an inhibitory effect on NPCs and neural differentiation in vitro.The number of neurospheres formed from primary NPCs significantlydecreased in the presence of either recombinant CCL11 (FIGS. 12B-12C) orCCL2 (FIG. 12D). Additionally, neurosphere size also decreased in thepresence of CCL11 (FIGS. 12E-12F). Using a human derived NTERA cell lineexpressing eGFP under the Doublecortin promoter, we assayed neuraldifferentiation and observed a significant decrease in eGFP expressionafter twelve days in culture with either CCL11 (FIG. 12G) or CCL2 (FIG.12H) under differentiation conditions. Our data demonstrate thatinhibitory factors present in aged blood are sufficient to act directlyon NPCs in vitro. While these findings, together with studies showing alack of a classical BBB in the neurogenic niche 13-15, open thepossibility of a direct interaction of systemic factors with progenitorcells in vivo during aging, they do not preclude the possibility thatage-related systemic factors may also act indirectly by stimulatingother cell types that comprise the neurogenic niche to releaseadditional inhibitory factors.

To examine the direct effect of CCL11 on neurogenesis in the brain, westereotaxically injected recombinant CCL11 into the DG of young adultmice, and observed a decrease in the number of Dcx-positive cells whencompared with the contralateral DG receiving vehicle control (FIG. 13).Furthermore, as an additional test of direct actions of systemic factorsin the brain, we examined whether the inhibitory effect of peripheralCCL11 on neurogenesis could be restored locally by inhibiting CCL11action specifically within the hippocampus. To test this, westereotaxically injected CCL11-specific neutralizing antibody into theDG and isotype control antibodies into the contralateral DG of youngadult mice. Following stereotaxic injection, we systemicallyadministered either recombinant CCL11 or vehicle control byintraperitoneal injections. The decrease in Dcx-positive cell numberobserved in animals receiving systemic CCL11 administration could berescued by neutralizing CCL11 within the DG with antigen specificantibodies but not isotype controls (FIGS. 4E-4F), suggesting thatincreases in systemic chemokine levels exert a direct effect in the CNS.

Finally, to determine the physiological relevance of increased systemicCCL11 levels in mice we assessed hippocampal dependent learning andmemory using the RAWM paradigm (FIG. 2D). Cohorts of young adult micereceived intraperitoneal injections of recombinant murine CCL11 or PBSvehicle as a control. All mice showed similar spatial learning capacityduring the training phase regardless of treatment (FIG. 4G). However, bythe end of the testing phase animals receiving recombinant CCL11 proteinexhibited impaired learning and memory deficits, committingsignificantly more errors in locating the target platform than animalsreceiving vehicle control (FIG. 4G). Together, these functional datademonstrate that increasing the systemic level of CCL11 not only inhibitadult neurogenesis but also impair hippocampal dependent learning andmemory.

Cumulatively, our data link age-related molecular changes in thesystemic milieu to the age-related decline in adult neurogenesis andassociated impairments in synaptic plasticity and cognitive functionobserved during aging (FIGS. 14A-14B). We demonstrate that the influenceof the aging systemic milieu is significant, and one that changes in anage-dependent fashion, potentially contributing to the susceptibility ofthe aging brain to cognitive impairments. The proteomic platform we usedhere was suitable to identify age-related systemic factors which inhibitadult neurogenesis.

We now show that an increase in the systemic levels of immune-relatedfactors present in old blood is capable of diminishing adultneurogenesis and impairing spatial learning and memory. We identifiedage-related chemokines classically involved in peripheral inflammatoryresponses as biologically relevant inhibitory factors of neurogenesis incell culture and in the CNS. Interestingly, CCL2, CCL11 and CCL12 arelocalized to within 70 kB on mouse chromosome 11, and likewise, CCL2 andCCL11 are within 40 kB on human chromosome 17 (mouse CCL12 is ahomologue of human CCL2 and does not exist in humans), implicating thisgenetic locus in normal brain aging and possibly aging in general.Indeed, work investigating cellular senescence, a known hallmark ofaging, furthers the involvement of some of the individual systemicchemokines reported here (CCL2) in the aging process as components ofthe Senescence-Associated Secretory Phenotype (Fumagalli, M. & d'Adda diFagagna, F., Nat Cell Biol 11 (8), 921-923 (2009)).

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofthe present invention is embodied by the appended claims.

1. A method of treating an adult mammal for an aging-associatedimpairment, the method comprising: modulating CCR3 in the mammal in amanner sufficient to treat the adult mammal for the aging-associatedimpairment.
 2. The method according to claim 1, wherein modulating CCR3comprises modulating eotaxin-1/CCR3 interaction.
 3. The method accordingto claim 2, wherein eotaxin-1/CCR3 interaction is modulated by reducingactive systemic eotaxin-1 in the mammal.
 4. The method according toclaim 3, wherein the active systemic eotaxin-1 is reduced in the mammalby administering to the mammal an effective amount of an active systemiceotaxin-1 reducing agent.
 5. The method according to claim 4, whereinthe active systemic eotaxin-1 reducing agent is an eotaxin-1 bindingagent.
 6. The method according to claim 5, wherein the eotaxin-1 bindingagent comprises an antibody or binding fragment thereof.
 7. The methodaccording to claim 5, wherein the eotaxin-1 binding agent comprises asmall molecule.
 8. The method according to claim 4, wherein the activesystemic eotaxin-1 reducing agent comprises an eotaxin-1 expressioninhibitory agent.
 9. The method according to claim 8, wherein theeotaxin-1 expression inhibitory agent comprises a nucleic acid.
 10. Themethod according to claim 1, wherein eotaxin-1/CCR3 interaction ismodulated by reducing CCR3 activity in the mammal.
 11. The methodaccording to claim 10, wherein the CCR3 activity is reduced in themammal by administering to the mammal an effective amount of an activeCCR3 reducing agent.
 12. The method according to claim 11, wherein theactive CCR3 reducing agent is a CCR3 binding agent.
 13. The methodaccording to claim 12, wherein the CCR3 binding agent comprises anantibody or binding fragment thereof.
 14. The method according to claim12, wherein the CCR3 binding agent comprises a small molecule.
 15. Themethod according to claim 11, wherein the active CCR3 reducing agentcomprises a CCR3 expression inhibitory agent.
 16. The method accordingto claim 15, wherein the CCR3 expression inhibitory agent comprises anucleic acid.
 17. The method according to claim 1, wherein the mammal isa primate.
 18. The method according to claim 17, wherein the primate isa human.
 19. (canceled)
 20. The method according to claim 1, wherein theelderly mammal is a human that is 60 years or older.
 21. The methodaccording to claim 1, wherein the aging-associated impairment comprisesa cognitive impairment. 22-23. (canceled)
 24. The method according toclaim 1, wherein the adult mammal suffers from an aging associateddisease condition.
 25. The method according to claim 1, wherein theaging associated disease condition is a cognitive decline diseasecondition.